Method and apparatus for processing tissue samples

This application is a continuation of U.S. patent application Ser. No. 18/119,810 filed Mar. 9, 2023, which is a continuation of U.S. patent application Ser. No. 15/734,128, filed Dec. 1, 2020, (now U.S. Pat. No. 11,618,876, issued Apr. 4, 2023), which is a U.S. 371 national stage entry of PCT/US19/35097 filed Jun. 1, 2019, which claims the benefit of the priority date of U.S. provisional patent application No. 62/679,494, filed Jun. 1, 2018, which applications are incorporated herein by reference in their entireties.

This research was supported in part by the National Human Genome Research Institute of the National Institutes of Health under award number RH010129. The government has certain rights in this invention.

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This invention relates to the field of sample preparation from biological materials. More specifically, the invention relates to the processing of solid tissues into single cells, nuclei, biomolecules, and processed samples for bioanalysis and the use of single cells to form organoids and other microtissues.

Analysis of single cells and groups of cells is providing information to dissect and understand how cells function individually and unprecedented insight into the range of individual cellular responses aggregated in ensemble measurements. Single cell methods for electrophysiology, flow cytometry, imaging, mass spectrometry (Lanni, E. J., et. al. J Am Soc Mass Spectrom. 2014; 25(11):1897-907.), microarray (Wang L and KA Janes. Nat Protoc. 2013; 8(2):282-301.), and Next Generation Sequencing (NGS) (Saliba A. E., et. al. Nucleic Acids Res. 2014; 42(14):8845-60.) have been developed and are driving an increased understanding of fundamental cellular processes, functions, and interconnected networks. As the individual processes and functions are understood and differentiated from ensemble measurements, the individual information can in turn lead to discovery of how network processes among cells operate. The networks may be in tissues, organs, multicellular organisms, symbionts, biofilms, surfaces, environments, or anywhere cells live and interact.

Model systems of tissues are an important tool in the understanding of tissue function, development, and regulation. Improved model systems are needed to be developed for basic research, companion diagnostics, and screening of compounds to develop therapeutics. Historically, two dimensional culture of model cell lines have been used to gain knowledge and model tissue function, typically with only one cell type at a time. However, solid tissues are three dimensional (3D) structures with complex interactions between multiple cell types. The two dimensional model can lack cell to cell interactions and the impacts of the extracellular matrix on the cells.

3D structures of tissues, which can be termed microtissues, can be constructed using many techniques including hanging droplets or using 3D supports comprised of Matricel or hydrogels to create small spheroid and other shapes that can better mimic actual tissues. Typically the size is limited by the diffusion of nutrients into the 3D structure. In some cases, the 3D is created incorporating stem cells and can be termed an organoid (N. de Souza, Organoids. Nature Methods volume 15, page 23 (2018); “Method of the Year 2017: Organoids” Nature Methods 2018/01/03/online, 15 http://dx.doi.org/10.1038/nmeth.4575.10.1038/nmeth.4575; Yin, Xiaolei et al. Engineering Stem Cell Organoids, Cell Stem Cell, Volume 18, Issue 1, 25-38). While organoids are a promising technology, the creation of organoids suffers from process variablity and the use of cells typically not derived directly from tissue. This invention solves the problems of releasing cells from solid tissues for multiple applications including growing cells on 2D surfaces, 3D organoids, and cell suspensions.

Next Generation Sequencing (NGS) of single cells is rapidly changing the state of knowledge of cells and tissue, discovering new cell types, and increasing understanding of the diversity of how cells and tissue function. Single cell NGS RNA sequencing (Saliba A. E., et. al., Nucleic Acids Res. 2014; 42(14):8845-60.) (Shapiro E. et. al., Nat Rev Genet. 2013; 14(9):618-30.) is unveiling the complexity of cellular expression, and the heterogenity from cell to cell, and from cell type to cell type (Buettner F. et. al., Nat Biotechnol. 2015; 33(2):155-60.). In situ sequencing (Ke R et. al., Nat Methods. 2013; 10(9):857-60.), (Lee J H, et. al., Nat Protoc. 2015; 10(3):442-58.) (Lee J H, et. al., Science. 2014, 21; 343(6177):1360-3.) has shown the feasability of directly sequencing fixed cells. However, for RNA, many fewer reads are generated with in situ sequencing, biasing against detection of low abundant transcripts. Photoactivatable tags have been used to capture mRNA from single cells (Lovatt, D., et. al., Nat Methods. 2014; 11(2):190-6.) from known locations in tissue, albeit with low throughput capture and manual cell collection.

The NGS market has grown explosively over the last 10 years with cost reductions and throughput increases exceeding Moore's law. The applications have expanded from whole genome sequencing to RNA-Seq, ChIP-Seq, exome sequencing, to now single-cell sequencing, single nuclei sequencing, ATAC-Seq, and many other exciting applications. The power and low cost of NGS is broadly changing life sciences and moving into translational medicine and the clinic as precision medicine begins. Until recent years essentially all of the NGS analysis was of ‘bulk samples’ where the nucleic acids of numerous cells had been pooled. There is a need for systems that integrate the sample preparation of single-cell suspensions, and single-cell libraries, and bulk libraries starting from original unprocessed fresh specimens as well as banks of frozen tissue and Formalin-Fixed Paraffin-Embedded (FFPE) tissue. This instant invention enables a system, cartridges, and processes to process solid tissues from many types of specimens comprising single cells, single nuclei, and nucleic acids.

Single-cell sequencing is rapidly changing the state of knowledge of cells and tissue, discovering new cell types, and increasing the understanding of the diversity of how cells and tissue function. Single-cell RNA sequencing (Shapiro E. Biezuner T, Linnarsson S. Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat Rev Genet. 2013; 14(9):618-30. PMID: 23897237) has highlighted the complexity of cellular expression, and the large heterogeneity from cell-to-cell, and from cell type-to-cell type (Buettner F. Natarajan K N, Casale F P, Proserpio V, Scialdone A, Theis F J, Teichmann S A, Marioni J C, Stegle O. Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells. Nat Biotechnol. 2015; 33(2):155-60. PMID: 25599176). Single-cell sequencing (Wang., Y. and N. E. Navin. Advanced and Applications of single-cell sequencing technologies. Molecular Cell. 2015. 58:598-609. PMID 26000845.) is being applied to development, brain structure and function, tumor progression and resistance, immunogenetics, and more.

Single cell nucleic acid sequencing technology and methods using NGS and Next Next Generation Sequencing (NNGS), such as nanopores, are rapidly evolving. Common components are incorporation of a marker or barcode for each cell and molecule, reverse transcriptase for RNA sequencing, amplification, and pooling of sample for NGS and NNGS (collectively termed NGS) library preparation and analysis. Starting with isolated single cells in wells, barcodes for individual cells and molecules have been incorporated by reverse transcriptase template switching before pooling and polymerase chain reaction (PCR) amplification (Islam S. et. al. Genome Res. 2011; 21(7):1160-7.) (Ramskold D. et. al. Nat Biotechnol. 2012; 30(8):777-82.) or on a barcoded poly-T primer with linear amplification (Hashimshony T. et. al. Cell Rep. 2012 Sep. 27; 2(3):666-73.) and unique molecular identifiers (Jaitin D. A. et. al. Science. 2014; 343(6172):776-9.).

Pioneering work has used micronozzles (Geng T. et. al. Anal Chem. 2014; 86(1):703-12) to produce nanodroplets to perform highly parallel processing of mRNA from single cells with reverse transcription incorporating cell and molecular barcodes from freed primers (inDrop) (Klein A. M. et. al. Cell. 2015; 161(5):1187-201.) or primers attached to paramagnetic beads (DropSeq) (Macosko E. Z. et. al. Cell. 2015; 161(5):1202-14.); the lysis conditions and reverse transcriptase described by (Fekete R. A. and A. Nguyen. U.S. Pat. No. 8,288,106. Oct. 16, 2012) are incorporated by reference cited therein are incorporated by reference, including instrumentation, chemistry, workflows, reactions conditions, flowcell design, and other teachings. Both inDrop and DropSeq are scalable approaches have change the scale from 100 s of cells previously analyzed to 1,000 s and more.

Single-cell sequencing is now providing new information to biologists, genomic scientists, and clinical practitioners, and the single-cell market is growing explosively, perhaps the next great disruption in life sciences and medicine. Multiple companies are providing systems to take single-cell suspensions and create Single-cell RNA sequencing (scRNA-Seq) libraries that are analyzed by the robust NGS sequencing and analysis pipeline. No system integrates the upstream process to produce single-cell suspensions for NGS single-cell sequencing or has integrated from tissue to single-cell or single nuclei libraries.

The production of single-cells or nuclei or nucleic acids from solid and liquid tissue is usually performed manually with a number of devices used without process integration. A combination of gentle mechanical disruption with enzymatic dissociation has been shown to produce single-cells with the highest viability and least cellular stress response (Quatromoni J G, Singhal S, Bhojnagarwala P, Hancock W W, Albelda S M, Eruslanov E. An optimized disaggregation method for human lung tumors that preserves the phenotype and function of the immune cells. J Leukoc Biol. 2015 January; 97(1):201-9. doi: 10.1189/jlb.5TA0814-373. Epub 2014 Oct. 30.).

Many manual protocols for dissociating different tissues exist, for example, Jungblut M., Oeltze K., Zehnter I., Hasselmann D., Bosio A. (2009). Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator. JoVE. 29, doi: 10.3791/1266; Stagg A J, Burke F, Hill S, Knight S C. Isolation of Mouse Spleen Dendritic Cells. Protocols, Methods in Molecular Medicine. 2001: 64: 9-22. Doi: 10.1385/1592591507.; Lancelin, W., Guerrero-Plata, A. Isolation of Mouse Lung Dendritic Cells. J. Vis. Exp. (57), e3563, 2011. DOI: 10.3791/3563; Smedsrod B, Pertoft H. Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence. J Leukocyte Biol. 1985: 38: 213-30.; Meyer J, Gonelle-Gispert C, Morel P, Buhler L Methods for Isolation and Purification of Murine Liver Sinusoidal Endothelial Cells: A Systematic Review. PLoS ONE 11(3) 2016: e0151945. doi:10.1371/journal.pone.0151945.; Kondo S. Scheef E A, Sheibani N, Sorenson C M. “PECAM-1 isoform-specific regulation of kidney endothelial cell migration and capillary morphogenesis”, Am J Physiol Cell Physiol 292: C2070-C2083, (2007); doi: 10.1152/ajpcell.00489.2006.; Ehler, E., Moore-Morris, T., Lange, S. Isolation and Culture of Neonatal Mouse Cardiomyocytes. J. Vis. Exp. (79), e50154, doi:10.3791/50154 (2013).; Volovitz I Shapira N, Ezer H, Gafni A, Lustgarten M, Alter T, Ben-Horin I, Barzilai O, Shahar T, Kanner A, Fried I, Veshchev I, Grossman R, Ram, Z. A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single cells. BMC Neuroscience (2016) 17:30 doi: 10.1186/s12868-016-0262-y; F. E Dwulet and M. E. Smith, “Enzyme composition for tissue dissociation,” U.S. Pat. No. 5,952,215, Sep. 14, 1999.

For example, solid tissue of interest is usually dissected and then minced into 1-5 mm pieces by hand or a blender type of disruptor is used. Enzymes or a mixture of enzymes, such as collagenases, hydrauronadase, papain, proteases, DNase, etc., are added and the specimen incubated, typically with shaking or rotation to aid dissociation to prepare single cells or nuclei from tissue. In many procedures, the specimen is triturated multiple times or mechanically disrupted. The mechanical disruption may be through orifices, grinding, homogenization, forcing tissue through screens or filters, sonication, blending, bead-beating, rotors with features that dissociate tissue, and other methods to physically disrupt tissue to help produce single cells.

Following dissociation, in some embodiments the dissociated sample is passed through a filter, such as a 70 μm filter, to retain clumps of cells or debris. The filtrate which contains single cells or nuclei may be further processed to change the media or buffer; add, remove, or deactivate enzymes; concentrate cells or biomolecules, lyse red blood cells, or capture specific cell types. The processing typically involves multiple steps of centrifugation and resuspension, density gradients, or magnetic bead capture of specific cell types using antibodies, or other affinity capture ligands, or fluorescent cell-activated sorting (FACS), or other methods. The titer and viability of the single-cell suspension is usually determined using optical imaging with a microscope and haemocytometer, or an automated instrument. In many cases, the viability is determined using Trypan blue or fluorescent dyes. Quality control can include characterization of the nucleic acids by gel electrophoresis on an instrument such as a BioAnalyzer, or the determination of the expression of certain genes using reverse transcripatase and quantitative polymerase chain reaction (RT-qPCR), or other relevant methods.

The rapid production of nuclei can give a snapshot of gene expression (Habib N, Li Y, Heidenreich M, Swiech L, Avraham-Davidi I, Trombetta J J, Hession C, Zhang F, Regev A. Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons. Science. 2016 Aug. 26; 353(6302):925-8. doi: 10.1126/science.aad7038. Epub 2016 Jul. 28.; Grindberg R V, Yee-Greenbaum J L, McConnell M J, Novotny M, O'Shaughnessy A L, Lambert G M, Arauzo-Bravo M J, Lee J, Fishman M, Robbins G E, Lin X, Venepally P, Badger J H, Galbraith D W, Gage F H, Lasken R S. RNA-sequencing from single nuclei. Proc Natl Acad Sci USA. 2013 Dec. 3; 110(49):19802-7. doi: 10.1073/pnas.1319700110. Epub 2013 Nov. 18.).

The production of nuclei from tissue can be performed using a Dounce homogenizer in the presence of a buffer with a detergent that lyses cells but not nuclei. Nuclei can also be prepared starting from single cell suspensions (CG000124_SamplePrepDemonstratedProtocol_-_Nuclei_RevB, 10× Genomics, https://assets.contentful.com/an68im79xiti/6FhJX6yndYy0OwskGmMc8l/48c341c178fe afa3ce21f5345ed3367b/CG000124_SamplePrepDemonstratedProtocol_-_Nuclei_RevB.pdf) by addition of a lysis buffer such as 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2 and 0.005% Nonidet P40 in nuclease-free water and incubation for 5 min on ice before centifugation to pellet the nuclei followed by resuspension in a resuspension buffer such as 1× Phosphate Buffered Saline (PBS) with 1.0% BSA and 0.2 U/μl RNase Inhibitor. The nuclei may be repeatedly pelleted and resuspended to purify them or density gradients or other purification methods used. The titer and viability of the nuclei suspension is usually determined using optical imaging with a microscope and haemocytometer, or an automated instrument with viability determined using Trypan blue or fluorescent dyes.

The multi-process workflow to produce and characterize single-cells and nuclei from tissue is a usually performed manually using several devices without process integration, limiting the scalablity of single cell sequencing and the integration with downstream processes to create a sample-to-answer system. It is laborious and requires skilled technicians or scientists, and results in variability in the quality of the single-cells, and, therefore, in the downstream libraries, analysis, and data. The multiple steps and skill required can lead to differing qualities of single cells or nuclei produced even from the same specimen, limiting clinical utility. Today, the production of high quality single-cells can take months of optimization.

Standarization is necessary before routine single-cell preparation can be performed, particularly in clinical settings. In addition, the length of the process and the process of dissociation can lead to the tissue and cells changing physiology and altering their expression of biomolecules such as RNA, proteins, lipids, and metabolites in response to the stresses of the procedure, accentuated by potentially long processing times. A crucial recent insight is that cell processing methods, for example, the use of a protease to dissociate cells from tissue (Lacar B, Linker S B, Jaeger B N, Krishnaswami S, Barron J, Kelder M, Parylak S, Paquola A, Venepally P, Novotny M, O'Connor C, Fitzpatrick C, Erwin J, Hsu J Y, Husband D, McConnell M J, Lasken R, Gage F H. Nuclear RNA-seq of single neurons reveals molecular signatures of activation. Nat Commun. 2016 Apr. 19; 7:11022. doi: 10.1038/ncomms11022. PMID: 27090946.) can alter gene expression by placing cells under stress, confounding analysis of the true transcriptome.

Robust, automated sample preparation is required to simplify workflows before full process or physical integration with downstream NGS analysis can be achieved to produce true sample-to-answer solid tissue to single cell/nuclei NGS analysis systems in the future. Robust processes and automated systems are required that will input a wide range of tissues from a wide range of organisms and tissues and produce high-quality single-cell or nuclei suspensions without intervention, at acceptable viability for suspensions, with minimal changes to gene expression patterns.

To achieve a standardized process will require a system that automates the sample preparation of cells or nuclei from tissue with a single-use disposable cartridge. In some cases, microvalves can be used in cartridges. Microvalves are comprised of mechanical (thermopneumatic, pneumatic, and shape memory alloy), non-mechanical (hydrogel, sol-gel, paraffin, and ice), and external (modular built-in, pneumatic, and non-pneumatic) microvalves (as described in: C. Zhang, D. Xing, and Y. Li., Micropumps, microvalves, and micromixers within PCR microfluidic chips: Advances and trends. Biotechnology Advances. Volume 25, Issue 5, September-October 2007, Pages 483-514; Diaz-González M., C. Fernández-Sánchez, and A. Baldi A. Multiple actuation microvalves in wax microfluidics. Lab Chip. 2016 Oct. 5; 16(20):3969-3976.; Kim J., Stockton A M, Jensen E C, Mathies R A. Pneumatically actuated microvalve circuits for programmable automation of chemical and biochemical analysis. Lab Chip. 2016 Mar. 7; 16(5):812-9. doi: 10.1039/c5lc01397f; Samad M F, Kouzani A Z. Design and analysis of a low actuation voltage electrowetting-on-dielectric microvalve for drug delivery applications. Conf Proc IEEE Eng Med Biol Soc. 2014; 2014:4423-6. doi: 10.1109/EMBC.2014.6944605.; Samad M F, Kouzani A Z. Design and analysis of a low actuation voltage electrowetting-on-dielectric microvalve for drug delivery applications. Conf Proc IEEE Eng Med Biol Soc. 2014; 2014:4423-6. doi: 10.1109/EMBC.2014.6944605.; Lee E, Lee H, Yoo S I, Yoon J. Photothermally triggered fast responding hydrogels incorporating a hydrophobic moiety for light-controlled microvalves. ACS Appl Mater Interfaces. 2014 Oct. 8; 6(19):16949-55. doi: 10.1021/am504502y. Epub 2014 Sep. 25.; Liu X, Li S. An electromagnetic microvalve for pneumatic control of microfluidic systems. J Lab Autom. 2014 October; 19(5):444-53. doi: 10.1177/2211068214531760. Epub 2014 Apr. 17; Desai A V, Tice J D, Apblett C A, Kenis P J. Design considerations for electrostatic microvalves with applications in poly(dimethylsiloxane)-based microfluidics. Lab Chip. 2012 Mar. 21; 12(6):1078-88. doi: 10.1039/c21c21133e. Epub 2012 Feb. 3.; Kim J, Kang M, Jensen E C, Mathies R A Lifting gate polydimethylsiloxane microvalves and pumps for microfluidic control. Anal Chem. 2012 Feb. 21; 84(4):2067-71. doi: 10.1021/ac202934x. Epub 2012 Feb. 1; Lai H, Folch A. Design and dynamic characterization of “single-stroke” peristaltic PDMS micropumps. Lab Chip. 2011 Jan. 21; 11(2):336-42. doi: 10.1039/c01c00023j. Epub 2010 Oct. 19). The system embodiments described herein can operate cartridges with no valves, or valves can be incorporated into the cartridges to direct flow.

Fluidic connections between cartridges and the instrument fluidics can be achieved by the use of spring-loaded connectors and modular microfluidic connectors as taught by Jovanovich, S. B. et. al. Capillary valve, connector, and router. Feb. 20, 2001. U.S. Pat. No. 6,190,616 and Jovanovich; S. B. et. al. Method of merging chemical reactants in capillary tubes, Apr. 22, 2003, U.S. Pat. No. 6,551,839; and Jovanovich, S., I. Blaga, and R. McIntosh. Integrated system with modular microfluidic components. U.S. Pat. No. 7,244,961. Jul. 17, 2007. which are incorporated by reference and their teachings which describe the modular microfluidic connectors and details of modular microfluidic connectors, including their use as multiway valves, routers, and other functions including microfluidic circuits to perform flowthrough reactions and flow cells with internally reflecting surfaces.

The surface chemistries of the paramagnetic beads and conditions to bind cells or precipitate, wash, and elute nucleic acids and other biomolecules onto surfaces is well understood, (Boom, W. R. et. al. U.S. Pat. No. 5,234,809. Aug. 10, 1993.), (Reeve, M. and P. Robinson. U.S. Pat. No. 5,665,554. Sep. 9, 1997.), (Hawkins, T. U.S. Pat. No. 5,898,071. Apr. 27, 1999.), (McKernan, K. et. al. U.S. Pat. No. 6,534,262. Mar. 18, 2003.), (Han, Z. U.S. Pat. No. 8,536,322. Sep. 17, 2013.), (Dressman et al., “Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variation” Proc. Natl. Acad. Sci. 100(15):8817-8822 (2003)), (Ghadessy et al., “Directed evolution of polymerase function by compartmentalized self-replication”, Proc. Natl. Acad. Sci. 98(8):4552-4557 (2000)), (Tawfik and Griffiths, “Man-made cell-like compartments for molecular evolution” Nat. Biotech. 16(7):652-656 (1998)), (Williams et al., “Amplification of complex gene libraries by emulsion PCR” Nat. Meth. 3(7):545-550 (2006)), and many chemistries are possible and within the scope of the instant disclosure.

Disclosed herein is a Sample Processing System that processes original or processed samples for bioanalysis or for the development of microtissues or organoids. The Sample Processing System processes are comprised of enzymatic and mechanical disruption mechanisms with integrated fluidic processes. This invention enables, among other things, the implementation of a Sample Processing System that inputs solid, liquid, or gaseous samples including tissue or other biological samples, and processes the samples for bioanalysis and other analyses.

In some embodiments, the sample or specimen is a tissue specimen. The tissue can be from any source such as a human, animal, or plant tissue. Examples of tissues include, without limitation, a biopsy sample, a core biopsy sample, a fine needle aspirate, cellular conglomerate, an organ fragment, whole blood, bone marrow, a biofilm, or any other solid, semi-solid, gelatinous, frozen or fixed three dimensional or two dimensional cellular matrix of biological. In another embodiment the released nucleic acid is bound to a membrane, chip surface, bead, surface, flow cell, or particle. The term specimen is used to mean samples and tissue specimens.

In one embodiment the Sample Processing System is used for tissue processing. A Tissue Processing System embodiment can be implemented as a flexible, extensible system that can process solid or liquid tissue and other samples into single cells, nuclei, organelles, and biomolecules with mechanical and enzymatic or chemical processes to produce single cells in suspension, nuclei, subcellular components, and biomolecules such as macromolecules comprised of nucleic acids, comprised of DNA and RNA; proteins; carbohydrates; lipids; biomolecules with multiple types of macromolecules: metabolites; and other biological components, including natural products for bioanalysis in suspension, in solution, or attached to a surface. In some embodiments, the Tissue Processing System performs affinity or other purifications to enrich or deplete cell types, organelles such as nuclei, mitochondria, ribosomes, or other organelles, or extracellular fluids. In some embodiments the Tissue Processing System can perform NGS library preparation. In some embodiments, the Tissue Processing System processes tissue into single-cell libraries for sequencing including Sanger, NGS, NNGS and other nucleic acid sequencing technologies, protein sequencing, or protoeomics, or other analytical methods.

Disclosed herein are different embodiments of Sample Processing Systems that integrate two or more of the overall steps to take samples from specimens (i.e., tissue, biofilms, other multi-dimensional matrices with cells or viruses, liquids) and prepare single cell or nuclei in suspensions or on surfaces, or further process the specimens into biomolecules including macromolecules comprised of nucleic acids, comprised of DNA and RNA; proteins; carbohydrates; lipids; biomolecules with multiple types of macromolecules; metabolites; and other biological components, including natural products). In some embodiments specimen can be processed into NGS sequencing libraries, or fully integrated with an analytical system to produce a sample-to-answer systems such as a sample-to-answer genomic system.

In some embodiments the Sample Processing System can be integrated with downstream bioanalysis to create a sample-to-answer system. In a preferred embodiment of the Sample Processing System, a Tissue Processing System processing embodiment is integrated with a nucleic acid bioanalysis system to sequence nucleic acids from tissues. Integrated is used to mean the workflows directly interface or in other contexts that the physical system directly interfaces or is incorporated into a system, instrument, or device. In one embodiment, the Tissue Processing System is integrated with a nucleic acid sequencer to produce a sample-to-answer system.

In one embodiment the Tissue Processing System can be used to create microtissues or organoids directly in a cartridge using the hanging droplet method or other methods, or the output of the system can be used as the starting point for creating microtissues or organoids off of the cartridge.

The Sample Processing System can have multiple subsystems and modules that perform processing or analysis. In a preferred embodiment of the Sample Processing System, one or more cartridges performs one or more steps in the processing workflow. In some embodiments the cartridges have multiple processing sites such as processing chambers that can process more than one sample. In some embodiments a cap couples mechanical disruption on the cartridge from a Physical Dissociation Subsystem to the Enzymatic and Chemical Dissociation Subsystem in a processing chamber. In some embodiments reagents from an Enzymatic and Chemical Dissociation Subsystem are delivered to the cartridge by a Fluidic Subsystem to regions that are used as Pre-Processing Chambers and Processing Chambers to disrupt or dissociate the specimen and process the cells, subcellular components, and biomolecules for bioanalysis.

The addition of fluids can be controlled by a Fluidic Subsystem with the complete system controlled by software in a Control Subsystem which can include the user interface through a device comprised of monitor, embedded display, touch screen, or through audio commands through the system or an accessory devices such as a cell phone or microphone. In some instances the Control Subsytem can include interfaces to laboratory information management systems, other instruments, databases, analysis software, email, and other applications.

In some embodiments, the amount of dissociation is monitored at intervals during the dissociation and in some instances the viability determined during processing using a Measurement Subsystem. The degree of dissociation and/or viability can be determined inside the main dissociation compartment and/or in a separate compartment or channel, and/or in the external instrument.

In some embodiments, cell imaging solutions, such as cell type specific antibodies, stains, or other reagents, can be added to the tissue or single cells or nuclei for additional processing or imaging. The imaging can capture cells, subcellular structures, or histological or other data. In some embodiments the images can be analyzed to direct the operation and workflow of the Sample Processing System through decisions trees, hash tables, machine learning, or artificial intelligence.

In some embodiments, single cells or nuclei in suspension or on surfaces are further processed using magnetic bead or particle technologies using a Magnetic Processing module to purify or deplete cell types, nuclei, nucleic acids, or other biomolecules.

The term singulated cells is used to mean single cells in suspension or on a surface or in a well including a microwell or nanowell such that they can be processed as single cells. The term singulated cells is also used at times to encompass single nuclei.

In one embodiment, the specimen is added to a cartridge which performs both physical and enzymatic dissociation of the tissue. In some embodiments the Singulator System performs trituration and other physical dissociation modalities as a step or steps in the process of singulating cells. The physical dissociation modalities include passing the specimen through screens, filters, orifices, grinding, blending, sonication, smearing, bead beating, and other methods known to one skilled in the art to physically disrupt tissue to help produce single cells or nuclei or nucleic acids or other biomolecules.

In one embodiment, the specimen is added to a cartridge which performs both physical and chemical dissociation of the tissue into nuclei. In some embodiments the Singulator System performs trituration and other physical dissociation modalities as a step or steps in the process of producing nuclei suspensions. The physical dissociation modalities include passing the specimen through screens, filters, orifices, gaps, grinding, blending, sonication, smearing, bead beating, and other methods known to one skilled in the art to physically disrupt tissue to help produce nuclei or nucleic acids or other biomolecules when using chemical treatment of tissues.

In one embodiment, the Sample Processing System is a Singulator System embodiment. The Singulator System described can input raw, unprocessed samples, or other primary or secondary samples, and output single cells or nuclei ready for single cell or nuclei analysis or for additional processing, e.g., to purify specific cell types with antibodies or by cell sorting or growth, library preparation, or many other applications. A Singulator System embodiment dissociates single cells or nuclei from specimens such as tissue, blood, bodily fluid or other liquids or solids containing cells to produce single cells in suspensions or nuclei, or on surfaces, in matrices, or other output configurations. In a preferred Singulation System described embodiment, there is a cartridge that inputs tissue and/or other specimens and outputs single cells or nuclei, preferably of known titer in a buffer supplemented with media such as Hank's buffer with 2% fetal calf serum.

In some embodiments, the Sample Processing System, such as a Singulator System embodiment, uses enzymes to assist in the process of singulating cells including enzymes to preserve nucleic acids and prevent clumping. The enzymes and additives are comprised of but not limited to collagenases (e.g., collagenases type I, II, III, IV, and others), elastase, trypsin, papain, tyrpLE, hyaluronidase, chymotrypsin, neutral protease, pronase, liberase, clostripain, caseinase, neutral protease (Dispase®), DNAse, protease XIV, RNase inhibitors, or other enzymes, protease inhibitors, active site inhibitors, EDTA, EGTA, biochemicals, or chemicals such as Triton X-100, Nonidet P40, detergents, surfactants, etc. In other embodiments, different reagents or mixtures of reagents are applied sequentially to dissociate the biological sample or specimen into single-cell suspensions.

In some embodiments, the Sample Processing System, such as a Singulator System embodiment, uses chemicals, enzymes, or both to assist in the process of producing nuclei from solid tissue in a nuclei isolation solution, assist in tissue dissociation, to preserve nucleic acids, and to prevent clumping. The chemicals are comprised of but not limited to detergents, surfactants, non-ionic surfactants, Triton X-100, Tween, Brij, CHAPS, Nonidet P40, Igepal, glycosides, HEGA, MEGA, or digitonin; the enzymes are comprised of collagenases (e.g., collagenases type I, II, III, IV, and others), elastase, trypsin, papain, tyrpLE, hyaluronidase, chymotrypsin, neutral protease, pronase, liberase, clostripain, caseinase, neutral protease (Dispase®), DNAse, protease XIV, or other enzymes. In some embodiments inhibitors such as RNase inhibitors, protease inhibitors, active site inhibitors, or biochemicals that sequester or chelate ions essential for RNases, comprising EDTA or EGTA or sodium citrate, can be used in solutions. In some embodiments, spermine or spermidine or sodium butyrate, or sodium orthovanadate, or sodium fluoride are included in the nuclei isolation solution or in nuclei storage solutions. In other embodiments, different reagents or mixtures of reagents are applied sequentially to dissociate the biological sample or specimen into single-cell suspensions and then the single cells are processed into nuclei. In some embodiment, the viscosity of the solutions are increased using chemicals comprised of ficoll, or gylcerol, or dextran, or sucrose, or trehalose, or polyethylene glycol, or cellulose or other compounds to slow diffusion rates of RNases or DNases or other enzymes or compounds that degrade biomolecules. In some embodiments the counterions in the buffers are acetate.

In some embodiments the Singulator System produces cell suspensions of known titers and viability. In some embodiments the Singulator System produces nuclei suspensions of known titers and quality. In some embodiments the Singulator System monitors the viability and/or the amount of singulation of a sample and adjusts the treatment time and concentration of enzymes or other dissociation agents by monitoring of the dissociation, for example by the production of single cells or nuclei. The monitoring can be in real time, in intervals, or endpoints or any combinations thereof.

The Singulator System can in some embodiments select from sets of reagents to dissociate tissue and adjust according to production of single cells or viability of cells as monitored by the system, in some instances in real time, at intervals, or as an endpoint. The single-cell suspensions produced by the Singulator System can be used to generate cells with therapeutic application, e.g., re-grow new tissues and/or organs and/or organisms.

The Singulator System has advantages over existing technology and can produce single cells, nuclei, or biomolecules from tissue in an automated and standardized instrument that can in some embodiments process the specimens into NGS libraries or other preparations. The Singulator System will enable users, e.g., researchers, clinicians, forensic scientists, and many disciplines to perform identical processing on biosamples, reducing user variability, and throughput constraints of manual processing.

Embodiments of the Singulation System can prepare single-cells or nuclei or nucleic acids for analysis by methods comprised of DNA sequencing, DNA microarrays, RNA sequencing, mass spectrometry, Raman spectroscopy, electrophysiology, flow cytometry, mass cytometry, and many other analytical methods well known to one skilled in the art including multidimensional analysis (e.g., LC/MS, CE/MS, etc.). In addition, single-cell suspensions or on surfaces or matrices can be used to grow additional cells including genetically altered by methods such as CRISPR, engineered viral or nucleic acid sequences, in tissue culture, or to grow tissues or organs for research and therapeutic purposes.

The Singulator System embodiment described is compatible with commercially available downstream library preparation and analysis by both NGS and NNGS sequencers. The term NGS is used to connote either NGS or NNGS sequencers or sample preparation methods as appropriate. As contemplated herein, next generation sequencing or next-next generation sequencing refers to high-throughput sequencing, such as massivley parallel sequencing, (e.g., simultaneously (or in rapid succession) sequencing any of at least 1,000, 100,000, 1 million, 10 million, 100 million, or 1 billion polynucleotide molecules). Sequencing methods may include, but are not limited to: high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), next generation sequencing, Single Molecule Sequencing by Synthesis (SMSS) (Helicos), massively-parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Maxam-Gilbert or Sanger sequencing, primer walking, sequencing using PacBio, SOLiD, Ion Torrent, Genius (GenapSys), DNA nanoball sequencing (Complete Genomics), or nanopore (e.g., Oxford Nanopore, Roche) platforms and any other sequencing methods known in the art.

In another aspect provided herein is an apparatus, composition of matter, or article of manufacture, and any improvements, enhancements, and modifications thereto, as described in part or in full herein and as shown in any applicable Figures, including one or more features in one or more embodiment.

In another aspect provided herein is an apparatus, composition of matter, or article of manufacture, and any improvements, enhancements, and modifications thereto, as described in part of in full herein and as shown in any applicable Figures, including each and every feature.

In another aspect provided herein is a method or process of operation or production, and any improvements, enhancements, and modifications thereto, as described in part or in full herein and as shown in any applicable Figures, including one or more feature in one or more embodiment.

In another aspect provided herein is a method or process of operation or production, and any improvements, enhancements, and modifications thereto, as described in part or in full herein and as shown in any applicable Figures, including each and every feature.

In another aspect provided herein is a product, composition of matter, or article of manufacture, and any improvements, enhancements, and modifications thereto, produced or resulting from any processes described in full or in part herein and as shown in any applicable Figures.

In one embodiment the single-cell suspension is prepared for a bioanalysis module for downstream analysis including but not limited to sequencing, next generation sequencing, next next generation sequencing, protein sequencing, proteomic analysis, genomic analysis, gene expression, gene mapping, carbohydrate characterization and profiling, lipid characterization and profiling, flow cytometry, imaging, DNA or RNA microarray analysis, metabolic profiling, functional, or mass spectrometry, or combinations thereof.

In another aspect provided herein is a data analysis system that correlates, analyzes, stores, and visualizes the analytical information of a sample component such as its viability, degree of single cell or nuclei dissociation, with the processing step and measures the change over time, and/or amount of enyzmatic activity, and/or physical disruptions of the original biological specimen. In another aspect provided herein is a data analysis system that correlates, analyzes, stores, and visualizes the analytical information of a sample component and shares metadata of the sample with downstream or upstream laboratory information systems.

In another aspect provided herein is a data analysis system that correlates, analyzes, and visualizes the analytical information of a sample component such as its viability, degree of single cell or nuclei dissociation, with the processing step and measures the change over time, and/or amount of enyzmatic activity, and/or physical disruptions of the original biological specimen and adjusts the processing parameters from the analytical information.

The Singulator System is a novel platform that automates and standardizes the only portion of the single-cell NGS workflow that has not been automated. This will have broad impacts. Process standardization will be critical for comparison of data from lab to lab or research to researcher. The Human Cell Atlas project intends to freely share the multi-national results in an open database. However, with no standardization of the complete process, direct comparisons will greatly suffer from widely varying impacts of the first processing step of producing single-cells or nuclei from tissue. Additionally, when single-cell or nuclei sequencing becomes clinically relevant, the standardization and de-skilling of the production of single-cells or nuclei will be required to be performed by an automated instrument such as the Singulator System.

In another aspect, provided herein is a system comprising: (a) an instrument comprising: (i) one or more cartridge interfaces configured to engage a cartridge; (ii) a fluidic subsystem comprising: (1) one or more containers containing one or more liquids and/or gasses; (2) one or more fluid lines connecting the containers with fluid ports in the cartridge interface; and (3) one or more pumps configured to move liquids and/or gasses into and/or out of the fluid port(s); (iii) one or more mechanical subsystems comprising an actuator; (iv) optionally, one or more magnetic processing modules comprising a source of magnetic force, wherein the magnetic force is positioned to form a magnetic field in one or more processing chambers; (v) optionally, one or more measurement modules; (vi) optionally, one or more control subsystems comprising a processor and memory, wherein the memory comprises code that, when executed by the processor, operates the system; and (b) one or more cartridges, each engaged with one of the cartridge interfaces, wherein each cartridge comprises: (i) a sample inlet port; (ii) one or more cartridge ports communicating with the fluid ports in the cartridge interface; (iii) a preprocessing chamber communicating with the sample inlet port and with at least one cartridge port, and comprising a tissue disruptor configured for mechanical disruption of tissue, wherein the tissue disruptor engages with and is actuated by the actuator when the cartridge is engaged with the cartridge interface; (iv) a strain chamber communicating with the preprocessing chamber configured to separate cells and/or nuclei from disrupted tissue optionally combined with either the preprocessing or processing chambers; (v) a processing chamber communicating with the strain chamber, optionally communicating with one or more cartridge ports and configured to perform one or more processing steps on separated cells and/or nuclei; and (vi) optionally, one or more waste chambers fluidically connected with the processing chamber. In one embodiment the tissue disruptor comprises a grinder, a pestle or a variable orifice. In another embodiment the system further comprises a barcode reader. In another embodiment the system comprises a measurement module that performs optical imaging to measure titer, clumping, and/or viability of cells or nuclei or properties of biomolecules. In another embodiment the system comprises a measurement module and a control system, wherein the measurement module measures, and one or more time points, characteristics of a sample in the processing chamber, and control system comprises code that determines a state of the sample, e.g., viability or degree of single cell or nuclei dissociation, and that adjusts processing parameters. In another embodiment the system further comprises (c) one or more analysis modules, wherein an input port of the analysis module is in fluid communication with the processing chamber. In another embodiment the analysis module performs an analysis selected from one or more of: DNA or RNA sequencing, next generation DNA or RNA sequencing, next next generation DNA or RNA sequencing of nucleic acids and their adducts such as epigenetic modifications; nanopore sequencing of nucleic acids and their adducts, single cell DNA sequencing of nucleic acids and their adducts; single nuclei RNA sequencing of nucleic acids and their adducts; PCR, digital droplet PCR, qPCR, RT-qPCR; genomic analysis, gene expression analysis, gene mapping, DNA fragment mapping; imaging including optical and mass spectrometry imaging; DNA or RNA microarray analysis; fluorescent, Raman, optical, mass spectrometery and other detection modalities of nucleic acids acids and their adducts with and without labels; proteomic analysis, including fluorescent, Raman, optical, mass spectrometery, protein sequencing, and other detection modalities of proteins and peptides and their adducts and modifications; carbohydrate characterization and profiling including sequencing, fluorescent, Raman, optical, mass spectrometery, and other detection modalities of carbohydrates and their adducts and other covalent polymers; lipid characterization and profiling including sequencing, fluorescent, Raman, optical, mass spectrometery, and other detection modalities of lipids and their adducts and other covalent polymers; flow cytometry; characterization of cells and profiling including fluorescent, Raman, optical, mass spectrometery, and other detection modalities of cells and their adducts and other covalent polymers; metabolic profiling including sequencing, fluorescent, Raman, optical, mass spectrometery, and other detection modalities of metabolites and their adducts and other covalent polymers; functional analysis including protein protein interactions; bioinformatic analysis of cells, organelles, and biomolecules; and mass spectrometry and other analytical methods.

In another embodiment the cartridge interface comprises a means of positioning the cartridge in the instrument that engages the fluidic subsystem and the mechanical module and optionally is temperature controlled. In another embodiment the cartridge is disposable.

In another aspect provided herein is a method comprising: (a) providing a tissue sample to a preprocessing chamber; (b) automatically performing mechanical and enzymatic/chemical disruption of the tissue in the preprocessing chamber to produce disrupted tissue comprising released cells and/or nuclei and debris; (c) automatically moving the disrupted tissue into a strain chamber comprising a strainer and/or filter and separating the released cells and/or nuclei from the debris therein; and (d) automatically moving the released cells and/or nuclei into a processing chamber which can be combined with the strain chamber in a preferred embodiment. In another embodiment automatically moving further comprises performing at least one processing step on the released cells and/or nuclei in the processing chamber. In another embodiment processing comprises one or more automatically performed processes selected from: (I) lysing cells; (II) capturing cells; (III) isolating nucleic acid; (IV) isolating protein; (V) converting RNA into cDNA; (VI) preparing one or more libraries of adapter tagged nucleic acids; (VII) performing PCR or other DNA amplification methods; (VIII) isolating individual cells or individual nuclei in nanodrops or nanoboluses or nanowells or in media such as agarose that will limit diffusion of molecules of interest between cells or nuclei; and (IX) outputting released cells and/or nuclei into output vessels such as 8 well strip tubes, microtiter plates, Eppendorf tubes, a chamber in the cartridge, or other vessels capable of receiving cell suspensions. In another embodiment the method further comprises: automatically capturing the released cells and/or nuclei in the processing chamber by binding to magnetically attractable particles comprising moieties having affinity for the cells and/or nuclei and applying a magnetic force to the processing chamber to immobilize the captured cells and/or nuclei. In another embodiment the method further comprises: automatically monitoring cell and/or nuclei titer in the preprocessing chamber and, when the titer reaches a desired level, exchanging a dissociation solution used to dissociate the tissue for a buffer. In another embodiment the method further comprises automatically monitoring a bioassay in the preprocessing or processing chambers.

In another aspect provided herein is a cartridge comprising: (i) a sample inlet port; (ii) optionally one or more cartridge ports configured to communicate with fluid ports in a cartridge interface; (iii) a preprocessing chamber communicating with the sample inlet port and with at least one cartridge port, and comprising a tissue disruptor configured for mechanical disruption of tissue, wherein the tissue disruptor engages with and is actuated by the actuator when the cartridge is engaged with the cartridge interface; (iv) a strain chamber communicating with the preprocessing chamber configured to separate cells from disrupted tissue that can optionally be combined with the preprocessing or processing chambers; (v) a processing chamber communicating with the strain chamber, optionally communicating with one or more cartridge ports and configured to perform one or more processing steps on separated cells; and (vi) optionally, one or more waste chambers fluidically connected with the processing chamber. In another embodiment the cartridge further comprises a cap that opens and closes the sample inlet port. In another embodiment the cap comprises a tissue disruptor element that moves, for example, about rotationally and back and forth along an axis. In another embodiment the cartridge further comprises a holder. In another embodiment the cartridge further comprises a top piece and a bottom piece connected by collapsible element which allow the top piece and/or the bottom piece to move relative to the holder. In another embodiment the holder comprises a mesh screen. In another embodiment the cartridge further comprises a grinding element for grinding tissue in the preprocessing chamber. In another embodiment the cartridge further comprises an identifier, such as a barcode or other identification system that comprises information about the cartridge and/or its use. In another embodiment the cartridge further comprises a plunger configured to move slideably within the preprocessing chamber. In another embodiment, the cartridge has one or more valves.

In another aspect provided herein is a variable orifice device for disrupting tissue comprising: (a) a first container and a second container fluidically connected through a flexible tube comprising a lumen; (b) an adjustable clamp positioned to clamp the flexible tube, wherein adjusting the clamp alters the cross-sectional area of the lumen; and (c) one or more pumps or devices operatively coupled with the first and/or second containers configured to push liquid in one container through the flexible tubing into the other container. In another embodiment the adjustable clamp comprises an eccentric cam operatively coupled to a motor, wherein rotating the cam closes or opens the clamp.

In another aspect provided herein is a method for disrupting tissue comprising: (a) providing a variable orifice device comprising first container and a second container fluidically connected through a flexible tube comprising a lumen; (b) moving a sample comprising tissue from one of the containers through the flexible tube to another one of the containers; (c) decreasing the cross-sectional area of the lumen and moving the sample from one of the containers through the flexible tube to another one of the containers; (d) repeating step (c) one or more times to disrupt the tissue.

In another aspect provided herein is a system comprising: (a) an instrument comprising: (i) one or more cartridge interfaces, each configured to engage a cartridge and comprising one or more fluid ports; (ii) a fluidics subsystem comprising: (1) one or more sources of liquids and/or gasses; (2) one or more fluid lines communicating with the sources and with fluid ports in the cartridge interface; and (3) one or more pumps configured to move liquids and/or gasses from the sources into and/or out of the one or more fluid ports; (iii) a subsystem comprising an actuator to actuate a tissue disruptor in a cartridge engaged with a cartridge interface (e.g., a mechanical, pneumatic, electromagnetic, or hydraulic actuator); and (b) one or more cartridges, each engaged with one of the cartridge interfaces, wherein each cartridge comprises: (i) one or more cartridge ports communicating with the fluid ports in the cartridge interface; (ii) a preprocessing chamber comprising an opening and, positioned in the opening, a tissue disruptor configured for mechanical disruption of tissue, wherein the tissue disruptor engages with and is actuated by the actuator when the cartridge is engaged with the cartridge interface; and (iii) a processing chamber communicating with the preprocessing chamber, and with one or more cartridge ports and configured to collect a suspension of biological material from the preprocessing chamber. In one embodiment, the instrument further comprises: none, one or a plurality of valves configured to direct positive or negative pressure from the one or more pumps through fluid lines and/or the one or more containers connecting the one or more fluid lines to the fluid ports. In another embodiment, the instrument further comprises: a magnetic processing module comprising a source of a magnetic field, wherein the source is positioned to form a magnetic field in a processing chamber of an engaged cartridge. In another embodiment, the instrument further comprises: a measurement subsystem. In another embodiment, the instrument further comprises: a control subsystem comprising a processor, memory, and a local or remote database wherein the memory comprises code that, when executed by the processor, operates the system and can store information of the instrument processes or analytical results from the system in a database. In another embodiment, the instrument further comprises: a waste container communicating with the one or more pumps. In another embodiment, the instrument further comprises: a temperature subsystem configured to regulate temperature in a chamber of the cartridge. In another embodiment, the temperature subsystem comprises a temperature regulating element (e.g., a Peltier, a resistive heater, a circulating fluid), a controller to control the temperature-regulating element and a thermal transfer element that transfers heat from the temperature-regulating element to or from the cartridge chambers. In another embodiment, the temperature subsystem comprises a temperature regulating element (e.g., a Peltier, a resistive heater, a circulating fluid), a controller to control the temperature-regulating element and a thermal transfer element that transfers heat from the temperature-regulating element to or from the reagents and reagent containers. In another embodiment, the actuator comprises a drive head selected from slotted, phillips, quadrex, tri-wing, spanner and hex. In another embodiment, the at least one pump comprises a syringe pump. In another embodiment, the pump communicates through a fluid line with a fluid port in the cartridge interface that engages a cartridge port that communicates with the processing chamber, wherein vacuum applied through the fluid line pulls fluid from the preprocessing chamber into the processing chamber. In another embodiment, the pump communicates through a first fluid line with a container of fluid and with a second line with a fluid port in the cartridge interface that engages a cartridge port that communicates with the preprocessing chamber or the processing chamber, wherein negative pressure applied through the first fluid line pulls fluid from container and positive pressure applied through the second fluid line pushes the fluid into the preprocessing chamber or the processing chamber. In another embodiment, each cartridge interface further comprises a reagent inlet port that communicates with a port in the preprocessing chamber configured to introduce reagent directly into the prepossessing chamber. In another embodiment, the preprocessing chamber communicates with the processing chamber directly through a fluid line, or indirectly, through one or more fluid lines in the interface that communicate with ports in each of the preprocessing chamber and the processing chamber. In another embodiment, the preprocessing chamber comprises no direct cartridge ports. In another embodiment, the cartridge comprises no more than any of one, two, three or four ports communicating with the cartridge interface or with the environment. In another embodiment, the cartridge comprises a plurality of ports communicating with the cartridge interface or with the environment, wherein at least one port is covered by a cap. In another embodiment, the tissue disruptor comprises: (i) a sheath, (ii) a plunger configured to move slidably within the sheath and comprising a coupler to engage the actuator and a head for disrupting tissue, and (iii) a bias mechanism, e.g., spring, that biases the plunger toward a retracted position, i.e. wherein actuation is required to actuate the plunger to a forward position. In another embodiment, the plunger also can rotate around the longitudinal axis of the sheath. In another embodiment, the head has a circumference which, when the head moves within the preprocessing chamber, provides a gap between the head and a wall of the preprocessing chamber between about 25 microns and 400 microns, e.g., sufficient to allow cells or nuclei or microstructures of cells to pass between the head and the wall without rupturing. In another embodiment, the head comprises a disruption surface comprising raised features for disrupting tissue. In another embodiment, the plunger comprises a top side comprising a feature for engaging the actuator. In another embodiment, the tissue disrupter is seated on a seat, e.g., an annular seat, in the preprocessing chamber. In another embodiment, the tissue disrupter head comprises an annular bevel, and the seat in the preprocessing chamber is configured to mate with the bevel such that when the plunger is actuated toward the bottom of the preprocessing chamber, there is a defined annular gap between the head and a wall of the preprocessing chamber, and no gap or a defined minimum gap between the disruption surface of the head and the bottom of the preprocessing chamber. In another embodiment, the preprocessing chamber comprises a bottom surface comprising raised features for disrupting tissue. In another embodiment, the preprocessing chamber communicates with the processing chamber through a fluidic channel connecting a port in a side of the preprocessing chamber with a port in a top of the processing chamber. In another embodiment, the processing chamber further comprises a strainer (e.g., filter or a mesh screen) positioned to strain suspension of biological material entering the processing chamber from the preprocessing chamber. In another embodiment, the processing chamber communicates with a cartridge port configured such that when vacuum is applied to the cartridge port, liquid in the preprocessing chamber is pulled into the processing chamber. In another embodiment, the cartridge further comprises a vacuum trap fluidically connected with and positioned between the cartridge port with the processing chamber. In another embodiment, the processing chamber comprises a drain section and a more narrow slot section and wherein the processing chamber comprises a slanted floor configured to direct fluid in the drain section toward the slot section. In another embodiment, the processing chamber comprises a depression for collecting a suspension of biological material. In another embodiment, the processing chamber communicates with a cartridge port configured to introduce fluids into the processing chamber. In another embodiment, the processing chamber comprises a cover comprising a port that communicates through a fluidic channel with the preprocessing chamber. In another embodiment, the preprocessing chamber comprises a cover comprising a seal (e.g. a friable seal, or septum) that, when removed or opened (e.g., punctured), allows access to the processing chamber. In another embodiment, the processing chamber comprises a cover comprising a seal (e.g. a friable seal, or septum) that, when removed or opened (e.g., punctured), allows access to the processing chamber. In another embodiment, the cartridge further comprises: one or more waste chambers fluidically connected with the processing chamber. In another embodiment, the cartridge further comprises an identifier comprising information about the cartridge and/or its use (e.g., a barcode, an RFID, an EE PROM), and wherein the instrument comprises a reader for reading information in the identifier. In another embodiment, the one or more sources of liquids and/or gasses are comprised in a fluidic subsystem.

In another aspect provided herein is a method comprising: (a) providing a system as disclosed herein, wherein the preprocessing chamber comprises a tissue sample; (b) disrupting the tissue sample by using the actuator to actuate the tissue disrupter to produce a suspension of biological material; and (c) using the fluidic subsystem to move the suspension of biological material from the preprocessing chamber into the processing chamber. In one embodiment, the method further comprises: removing the suspension of biological material from the processing chamber. In another embodiment, the prepossessing chamber further comprises one or more enzymes for digesting extracellular matrix. In another embodiment, the prepossessing chamber further comprises one or more detergents for lysing cell membranes. In another embodiment, the prepossessing chamber further comprises liquid having a viscosity that slows the rate of degradation of RNA or other biomolecules during or after tissue disruption. In another embodiment, disrupting comprises positioning a disruption surface of the head a defined distance from a bottom surface of the preprocessing chamber and rotating the head to disrupt tissue in the preprocessing chamber. In another embodiment, disrupting comprises positioning a disruption surface of the head with respect to a bottom surface of the preprocessing chamber at a plurality of different gap distances and, at each gap distance, rotating the head. In another embodiment, at at least one gap distance at least some portion of the disruption head contacts some portion of the bottom surface. In another embodiment, the widest gap distance between a flat portion of the head surface and flat portion of the bottom of the chamber is no more than any of 6 mm, 5 mm 4 mm, 3 mm, 2 mm, 1 mm, 500 μm, 250 μm, 100 μm, 75 μm, 50 μm, 25 μm, 20 μm, 15 μm, 10 μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm. In another embodiment, the plurality of gap distances between a flat portion of the head surface and flat portion of the bottom of the chamber is any of 2, 3, 4, 5, 6, 7, 8, 9 or 10 and the largest gap distance is no is no more than any of 6 mm, 5 mm 4 mm, 3 mm, 2 mm, 1 mm, 500 μm, 250 μm, 100 μm, 75 μm, 50 μm, 25 μm, 20 μm, 15 μm, 10 μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 um. In another embodiment, the method comprises: disrupting tissue with the tissue disruptor; incubating the disrupted tissue with at least one enzyme that digests extracellular matrix; and disrupting the incubated tissue with the tissue disruptor. In another embodiment, the fluidic subsystem applies a vacuum to a cartridge port communicating with the processing chamber to move the suspension of biological material. In another embodiment, the cartridge further comprises a strainer and the suspension of biological material entering the processing chamber is strained to remove particulate matter, e.g., clumps of cells or nuclei or subcellular biomolecules. In another embodiment, the method further comprises, after moving the suspension of biological material, using the fluidics subsystem to introduce a liquid into the preprocessing chamber through a cartridge port and then using the fluidics subsystem to move the liquid into the processing chamber. In another embodiment, the method further comprises using the fluidics subsystem to introduce one or more liquids comprising one or more reagents through a cartridge port into the processing chamber. In another embodiment, the reagent comprises an enzyme or a particle comprising a binding agent (e.g., a binding agent directed against a target on a cell surface or a surface of a nucleus or surface of a virus, or other biological target). In another embodiment, the tissue comprises a target cell and the method further comprises: contacting the suspension of biological material in the processing chamber with solid particles comprising binding agents that bind to the target cells and sequester bound target cells within the suspension of biological material. In another embodiment, the method further comprises separating the bound target cells from the suspension. In another embodiment, the tissue is tumor tissue and the target cells are tumor infiltrating lymphocytes. In another embodiment, the target cells are stem cells or partially differentiated cells. In another embodiment, the method further comprises: determining the expression of one or more genes in cells, nuclei, mitochondria or other organelles of the suspension of biological material. In another embodiment, the one or more genes is a panel comprising a plurality of genes. In another embodiment, the panel comprises genes distinguishing a target cell type, e.g., hepatocytes, neurons, kidney glomerulus parietal cell, cardiomyocytes. In another embodiment, the panel comprises genes distinguishing a CRISPR modified target cell. In another embodiment, the panel comprises genes that are differentially expressed when cells experience stress, e.g., anoikis. In another embodiment, the method comprises preparing a suspension of biological material on each of a plurality of tissue samples under different tissue disruption conditions, and identifying conditions that produce cells or nuclei having a gene expression profile closest to or furthest away from that of cells in the pre-disrupted tissue sample. In another embodiment, the panel comprises one or more housekeeping genes, e.g., a gene constitutively expressed at a relatively constant level in cells regardless of cellular stress states, e.g., Actb, gapdh.

In another aspect provided herein is a cartridge comprising: (i) a preprocessing chamber comprising: (1) an opening and, positioned in the opening, a tissue disruptor configured for mechanical disruption of tissue, and (2) a preprocessing chamber port; and (ii) a processing chamber comprising a processing chamber port communicating with the preprocessing chamber port through a fluid line, and (iii) a cartridge port that communicates with the processing chamber, wherein a vacuum applied to the cartridge port pulls material from the preprocessing chamber into the processing chamber. In one embodiment, the cartridge port communicates with the processing chamber directly or through a vacuum trap.

In another aspect provided herein is a cartridge comprising: (i) a preprocessing chamber comprising an opening and, positioned in the opening, a tissue disruptor configured for mechanical disruption of tissue; (ii) a strain chamber comprising a strainer, wherein the strain chamber communicates with the preprocessing chamber; (iii) a first processing chamber communicating with the strain chamber; (iv) an optional second processing chamber communicating with the first processing chamber; (v) one or more cartridge ports communicating with the processing chamber and the second processing chamber if present.

In one embodiment, the cartridge further comprises: one or more waste chambers communicating with the first processing chamber and second processing chamber when present. In one embodiment, the first processing chamber comprises an element (e.g., a nozzle) configured to produce a hanging drop of liquid from the strain chamber.

In another aspect provided herein is a method of creating a microtissue comprising: (a) providing a cartridge of as described herein comprising a tissue; (b) disrupting the tissue with the tissue disruptor to produce a cell suspension; (c) straining the cell suspension with the strainer to produce strained cell suspension; and (d) forming a hanging drop from the strained cell suspension using the element. In one embodiment, the microtissue is an organoid. In another embodiment, the method further comprises: after forming the hanging drop, adding a liquid or gas to the processing chamber to support survival of the cells in the hanging drop.

In another aspect provided herein is a method of creating a microtissue comprising: (a) providing a cartridge of as described herein comprising a tissue; (b) disrupting the tissue with the tissue disruptor to produce a cell suspension; (c) straining the cell suspension with the strainer to produce strained cell suspension; (d) selecting stem cells from strained cell suspension; and (e) removing or growing the selected stem cells in the cartridge.

In another aspect provided herein is a method of creating a microtissue comprising: (a) providing a cartridge of as described herein comprising a tissue; (b) disrupting the tissue with the tissue disruptor to produce a cell suspension; (c) straining the cell suspension with the strainer to produce strained cell suspension; (d) differentiating cells from strained cell suspension into stem cells; and (e) growing or removing the differentiated stem cells in the cartridge.

In another aspect provided herein is a system comprising: (a) an instrument comprising: (i) one or more cartridge interfaces, each configured to engage a cartridge and comprising one or more fluid ports; (ii) a module comprising an actuator to actuate a tissue disruptor in a cartridge engaged with a cartridge interface (e.g., a mechanical, pneumatic, electromagnetic, or hydraulic actuator); and (b) one or more cartridges, each engaged with one of the cartridge interfaces, wherein each cartridge comprises: (i) a preprocessing chamber comprising an opening and, positioned in the opening, a tissue disruptor configured for mechanical disruption of tissue. In one embodiment the cartridge does not include any chambers other than the preprocessing chamber.

In another aspect provided herein is a cartridge comprising: (i) a preprocessing chamber comprising an opening and, positioned in the opening, a tissue disruptor configured for mechanical disruption of tissue.

In another aspect provided herein is a tissue disruptor comprising: (i) a sheath, (ii) a plunger configured to move slidably within the sheath and comprising a coupler to engage the actuator and a head for disrupting tissue, and (iii) a bias mechanism, e.g., spring, that biases the plunger toward a retracted position, i.e., wherein actuation is required to actuate the plunger to a forward position. In one embodiment, the sheath comprises a seater element adapted to seat the tissue disruptor on a seat. In another embodiment, the seater element comprises a flange adapted to sit on an annular ring. In another embodiment, the seater element comprises one or more tabs adapted to sit in one or more slots.

NGS, mass spectrometry, FACS, and other modern high-throughput analysis systems have revolutionized life and medical sciences. The progression of information has been from the gross level of organism, to tissue, and now to single cell analysis. Single cell analysis of genomic, proteomic including protein expression, carbohydrate, lipid, and metabolism of individual cells is providing fundamental scientific knowledge and revolutionizing research and clinical capabilities.

Specimen: The term “specimen,” as used herein, refers to an in vitro cell, cell culture, virus, bacterial cell, fungal cell, plant cell, bodily sample, or tissue sample that contains genetic material. In certain embodiments, the genetic material of the specimen comprises RNA. In other embodiments, the genetic material of the specimen is DNA, or both RNA and DNA. In certain embodiments the genetic material is modified. In certain embodiments, a tissue specimen includes a cell isolated from a subject. A subject includes any organism from which a specimen can be isolated. Non-limiting examples of organisms include prokaryotes, eukaryotes or archaebacteria, including bacteria, fungi, animals, plants, or protists. The animal, for example, can be a mammal or a non-mammal. The mammal can be, for example, a rabbit, dog, pig, cow, horse, human, or a rodent such as a mouse or rat. In particular aspects, the tissue specimen is a human tissue sample. The tissue specimen can be liquid, for example, a blood sample, red blood cells, white blood cells, platelets, plasma, serum. The specimen, in other non-limiting embodiments, can be saliva, a cheek, throat, or nasal swab, a fine needle aspirate, a tissue print, cerebral spinal fluid, mucus, lymph, feces, urine, skin, spinal fluid, peritoneal fluid, lymphatic fluid, aqueous or vitreous humor, synovial fluid, tears, semen, seminal fluid, vaginal fluids, pulmonary effusion, serosal fluid, organs, bronchio-alveolar lavage, tumors, frozen cells, or constituents or components of in vitro cell cultures. In other aspects, the tissue specimen is a solid tissue sample or a frozen tissue sample or a biopsy sample such as a fine needle aspirate or a core biopsy or a resection or other clinical or veterinary specimen. In still further aspects, the specimen comprises a virus, archae, bacteria, or fungus. The specimen can be an ex vivo tissue or sample or a specimen obtained by laser capture microdissection. The specimen can be a fixed specimen, including as set forth by U.S. Published Patent Application No. 2003/0170617 filed Jan. 28, 2003, or a FFPE specimen.

In some embodiments, the single cells can be analyzed further for biomolecules including one or more polynucleotides or polypeptides or other macromolecules. In some embodiments, the polynucleotides can include a single-stranded or double-stranded polynucleotide. In some embodiments, the polypeptide can include an enzyme, antigen, hormone or antibody. In some embodiments, the one or more biomolecules can include RNA, mRNA, cDNA, DNA, genomic DNA, microRNA, long noncoding RNA, ribosomal RNA, transfer RNA, chloroplast DNA, mitochondrial DNA, or other nucleic acids including modified nucleic acids and complexes of nucleic acids with proteins or other macromolecules.

It will be readily apparent to one of ordinary skill in the art that the embodiments and implementations are not necessarily inclusive or exclusive of each other and may be combined in any manner that is non-conflicting and otherwise possible, whether they be presented in association with a same, or a different, embodiment or implementation. The description of one embodiment or implementation is not intended to be limiting with respect to other embodiments and/or implementations. Also, any one or more function, step, operation, or technique described elsewhere in this specification may, in alternative implementations, be combined with any one or more function, step, operation, or technique described in the summary. Thus, the above embodiment and implementations are illustrative rather than limiting.

One version of tissue processing system is described in International patent application PCT/US2017/063811 filed Nov. 29, 2017 (WO 2018/102471) (Jovanovich, Chear, McIntosh, Pereira, and Zaugg, “Method and Apparatus for Processing Tissue Samples”), incorporated herein in its entirely for all purposes.

1 FIG. 50 101 1000 1050 6001 6002 1060 1070 1071 1072 1073 1074 1075 1076 1077 1070 1071 1078 1079 shows a Sample Processing Systemthat can input specimenand process them to produce biologicals such as single cellsor nuclei, microtissues, organoids, or other biocomponents comprised of subcellular components, and biomoleculessuch as macromoleculesand nucleic acids, comprised of DNAand RNA; proteins; carbohydrates; lipids; biomoleculeswith multiple types of macromolecules; metabolites; and other biological components, including natural productsfor bioanalysis.

2 FIG. 80 120 101 1000 1050 6001 6002 1060 1070 1071 1072 1073 1074 1075 1076 1077 1070 1071 1078 1079 shows a Tissue Processing Systemthat can input tissue specimensand other specimensand process them to produce biologicals such as single cellsor nuclei, microtissues, organoids, or other biocomponents comprised of subcellular components, and biomoleculessuch as macromoleculesand nucleic acids, comprised of DNAand RNA; proteins; carbohydrates; lipids; biomoleculeswith multiple types of macromolecules; metabolites; and other biological components, including natural productsfor bioanalysis.

3 FIG. 100 101 120 1000 1050 6001 6002 1060 1070 1071 1072 1073 1074 1200 Referring to, the Singulation Systemaccepts one or more specimensor tissue specimensand processes them to produce biologicals such as single cellsor nuclei, microtissues, organoids, or other biocomponents comprised of subcellular components, and biomoleculessuch as macromoleculesand nucleic acids, comprised of DNAand RNAand single cell librariesfor bioanalysis.

4 FIG. 100 200 120 101 1000 1050 1072 1200 1250 1210 1060 1070 1071 1072 1073 1074 6001 6002 200 300 400 500 600 700 1475 900 Referring to, in some embodiments, the Singulation Systemprocessing is performed in cartridge(s)in the system. Tissue specimensor other specimensare converted to single cells, nuclei, nucleic acids, single cell libraries, single nuclei libraries, bulk libraries, or other biocomponents comprised of subcellular components, or biomoleculessuch as macromoleculesand nucleic acids, comprised of DNAand RNA, or microtissues, or organoidsthrough the use of one or more cartridgeswith one or more of the Physical Dissociation Subsystem, the Enzymatic and Chemical Dissociation Subsystem, the Measurement Subsystem, the Fluidic Subsystem, the Control Subsystem, Temperature Subsystem, and the Magnetic Module.

300 The Physical Dissociation Subsystem(which can include a preprocessing chamber, a tissue disruptor and an actuator) can perform physical disruption by passing the specimen through orifices, grinding, rotating a rotor with or without features to dissociate tissue, moving a head with or without features to dissociate tissue, forcing tissue through filters or screens or mesh or strainers, moving a pestle or Dounce like element, sonication, blending, homogenization, bead beating, pressure, and other methods known to one skilled in the art to physically disrupt tissue to help produce single cells or nuclei.

400 The Enzymatic and Chemical Dissociation Subsystem(which can include or use a source of fluid (e.g., comprising one or more enzymes or chemicals) and portions of the fluidic subsystem and cartridge interface that deliver liquids to a preprocessing or other chambers) can perform enzymatic disruption by adding formulations of a reagents or mixture of components comprised of but not limited to collagenases (e.g., collagenases type I, II, III, IV, and others), elastase, trypsin, papain, hyaluronidase, chymotrypsin, neutral protease, clostripain, caseinase, neutral protease (Dispase®), DNAse, protease XIV, RNase inhibitors, DNAse inhibitors, or other enzymes, biochemicals, or chemicals such as EDTA, EGTA, protease inhibitors, buffers, acids, or base.

400 1000 412 2 2 2 2 In another aspect, the Enzymatic and Chemical Dissociation Subsystemcan perform chemical disruption or chemical and enzymatic disruption is by adding formulations of chemicals that might disrupt tissue or cellular integrity such as Triton X-100, Tween, Nonident P40, other surfactants or detergents, digitonin, or biomolecules or chemicals that can dissociate tissue into cells or produce nuclei or other organelles directly from tissues or from single cellsuspensions. Many different nuclei isolation solutionshave been developed, including NST buffer (146 mM NaCl, 10 mM Tris base at pH 7.8, 1 mM CaCl, 21 mM MgCl, 0.05% BSA, 0.2% Nonidet P-40) (L. Martelotto, T Baslan, J, Kendall, F. C Geyer, K. A Burke, L. Spraggon, S. Piscuoglio, K. Chadalavada, G. Nanjangud, C. Ng, P. Moody, S. D'Italia, L. Rodgers, H. Cox, A. da Cruz Paula, A. Stepansky, M. Schizas, H. Y. Wen, T. A King, L. Norton, B. Weigelt, J. B Hicks, and J. S. Reis-Filho. Whole-genome single-cell copy number profiling from formalin-fixed paraffin-embedded samples. Nat Med. 2017 March; 23(3): 376-385. doi:10.1038/nm.4279.) or Homogenization buffer (10 mM Tris pH 8.0, 250 mM sucrose, 25 mM KCl, 5 mM MgCl, 0.1% Triton-X 100, (v/v), 0.4 U/μL RNasin Plus RNase inhibitor (Promega), 1× protease inhibitor, 0.2 U/μL Superasin (ThermoFisher), 10 ng/mL Hoechst 33342, and 0.1 μM DTT) (Krishnaswami S R, Grindberg R V, Novotny M, Venepally P, Lacar B, Bhutani K, Linker S B, Pham S, Erwin J A, Miller J A, Hodge R, McCarthy J K, Kelder M, McCorrison J, Aevermann B D, Fuertes F D, Scheuermann R H, Lee J, Lein E S, Schork N, McConnell M J, Gage F H, Lasken R S. Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons. Nat Protoc. 2016 March; 11 (3):499-524. doi: 10.1038/nprot.2016.015. PMID: 26890679.), or nuclear homogenization buffer (10 mM HEPES-KOH, pH 7.9, 25 mM KCl, 1 mM EDTA, 2 M sucrose, 10% glycerol, 0.15 mM spermine, 0.5 mM spermidine, 10 mM NaF, 1 mM orthovanadate, 1 mM PMSF, 0.5 mM DTT, and 1× protease inhibitor cocktail (Sigma)) (Ling G, Waxman D J. Isolation of nuclei for use in genome-wide DNase hypersensitivity assays to probe chromatin structure. Methods Mol Biol. 2013; 977:13-9. doi: 10.1007/978-1-62703-284-1_2. PubMed PMID: 23436350; PubMed Central PMCID: PMC3815455.) or 0.1× Lysis Buffer (1 mM TrisHCl, pH7.4, 1 mM NaCl, 0.3 mM MgCl, 0.01% Tween-20, 0.01% Nonldent P40, 0.001% digitonin, 0.1% bovine serum albumin) (Demonstrated Protocol—Nuclei Isolation from Mouse Brain Tissue for Single Cell ATAC Sequencing, Rev A, 10× Genomics) or 1×NIB: (10 mM MES-KOH (pH 5.4), 10 mM NaCl, 10 mM KCl, 2.5 mM EDTA, 250 mM sucrose, 0.1 mM spermine, 0.5 mM spermidine, 1 mM DTT. (S. Sikorskaite, M.-L. Rajamäki, D. Baniulis, V. Stanys and J. PT Valkonen. Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families. Plant Methods 2013, 9:31 http://www.plantmethods.com/content/9/1/31).

413 2 2 Similarly many different nuclei storage solutionshave been developed including Nuclei Wash and Resuspension Buffer (1×PBS with 1% BSA and 0.2 u/mL RNase Inhibitor (Sigma-Aldrich 3335399001)) (Demonstrated Protocol—Isolation of Nuclei for Single Cell RNA Sequencing, Rev B, 10× Genomics), or nuclear storage buffer (20 mM Tris-HCl, pH 8.0, 75 mM NaCl, 0.5 mM EDTA, 50% (v/v) glycerol, 1 mM DTT, and 0.1 mM PMSF) (Ling G, Waxman D J. Isolation of nuclei for use in genome-wide DNase hypersensitivity assays to probe chromatin structure. Methods Mol Biol. 2013; 977:13-9. doi: 10.1007/978-1-62703-284-1_2. PubMed PMID: 23436350; PubMed Central PMCID: PMC3815455.) or nuclear storage buffer (20% glycerol, 20 mM HEPES-KOH (pH 7.2), 5 mM MgCl, 1 mM DTT) (S. Sikorskaite, M.-L. Rajamäki, D. Baniulis, V. Stanys and J. PT Valkonen. Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families. Plant Methods 2013, 9:31 http://www.plantmethods.com/content/9/1/31) or NSB (166.6 mM sucrose, 5 mM MgCl, 10 mM Tris buffer, pH 8.0) (Krishnaswami S R, Grindberg R V, Novotny M, Venepally P, Lacar B, Bhutani K, Linker S B, Pham S, Erwin J A, Miller J A, Hodge R, McCarthy J K, Kelder M, McCorrison J, Aevermann B D, Fuertes F D, Scheuermann R H, Lee J, Lein E S, Schork N, McConnell M J, Gage F H, Lasken R S. Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons. Nat Protoc. 2016 March; 11(3):499-524. doi: 10.1038/nprot.2016.015. PMID: 26890679.).

In other embodiments, different reagents or mixtures of reagents are applied sequentially to dissociate the biological sample or specimen into single cells or nuclei. The physical and enzymatic/chemical dissociation and other subsystems can be separate from each other, or they can be co-located (e.g., acting upon the sample simultaneously or sequentially). The preprocessing, strain, and processing chambers can be separate from each other, or they can be co-located (e.g., acting upon the sample simultaneously or sequentially).

500 500 500 In some embodiments, the amount of dissociation is monitored at intervals during the dissociation or at the endpoint, and in some instances the viability is determined during processing using a Measurement Subsystem. The Measurement Subsystemcan be an optical imaging device to image cells using brightfield, phase contrast, fluorescence, chemiluminescence, near-field, Raman, or other optical readouts, or an optical measurement, or an electrical measurement, such as an impedance measurement of the change in conductivity, when a cell passes through a sensor, or thermal, or other types of measurement. In other embodiments Measurement Subsystemcan be a mass spectrometer, mass cytometer, or other system that determines mass.

600 600 The addition and movement of fluids can be performed by a Fluidic Subsystem. The Fluidic Subsystemcan use pumps, such as syringe pumps, piezopumps, electroosmotic pumps, peristalic pumps, on-cartridge pumps and valves, micropumps, pressure, pneumatics, or other components well known to one skilled in the art.

100 700 740 730 700 700 725 The Singulation Systemcan be controlled by software in a Control Subsystemwhich can be comprised of a user interfacethrough a monitor, embedded display, or a touch screen. In some instances the Control Subsytemcan include interfaces to laboratory information management systems, other instruments, analysis software, display software, databases, email, and other applications. The Control Subsystemcan include control softwareand scripts that control the operation and in some embodiments the scripts can be revised, created, or edited by the operator.

100 1475 200 1475 The Singulation Systemcan have temperature subsystemfor temperature regulation that can set the temperature of various parts of the system such as at reagent storage, or in fluidic lines, or in cartridge. The temperature subsystemcan use heating and or cooling from devices comprised of resistive heaters, Peltiers, circulating fluids, or other methods well known to one skilled in the art, with a temperature sensing element, such as a thermistor, thermocouple, thermoresponse color change, etc., and a temperature control board.

101 200 200 500 6002 6001 In another aspect provided herein is a device for the dissociation of a biological sample, the device comprising: (i) a biological sample or specimen; (ii) a cartridgecapable of dissociating tissue; (iii) an instrument to operate the cartridgeand provide fluids as needed (iv) a measurement modulesuch as an optical imaging to measure titer, clumping, and/or viability, or realtime PCR, (v) exchange of dissociation solution for buffer or growth media at the desired titer, and (vi) output vessels such as a chamber in the cartridge, 8 well strip tubes, microtiter plates, Eppendorf tubes, nanowells, or other vessels capable of receiving cell suspensions or an organoidor microtissue.

1000 1050 1010 1200 1210 1070 1000 1050 1200 1210 In another aspect provided herein is a device for the dissociation of a biological sample and the production of single-cellor nucleisuspensions or matched bulk nucleic acidsor single cell librariesor matched bulk libraries, the device comprising: (i) a chamber or area to input a biological sample or specimen; (ii) a cartridge capable of dissociating tissue or specimen; (iii) an instrument to operate the cartridge and provide fluids as needed (iv) a measurement module such as an optical imaging to measure titer, clumping, and/or viability, or the quantity of one or more biomolecules, (v) exchange of dissociation solution for buffer or growth media at the desired titer, (vi) the production of single-cellor nucleisuspensions or single cell libraries, and matched bulk nucleic acid libraries, in output vessels such as 8 well strip tubes, microtiter plates, Eppendorf tubes, a chamber in the cartridge, or other vessels capable of receiving cell suspensions.

4 FIG. 900 1000 1050 1060 1070 1071 1072 1073 1074 1075 1076 1077 1070 1071 1078 1079 1072 1050 Still referring to, a Magnetic Processing modulecan use magnetic processing of magnetic and paramagnetic particles or surfaces or beads, referred to as beads, to separate single cells, or cell types or nuclei, or other biocomponents comprised of subcellular components, and biomoleculessuch as macromoleculesand nucleic acids, comprised of DNAand RNA; proteins; carbohydrates; lipids; biomoleculeswith multiple types of macromolecules; metabolites; and other biological components, including natural productsfor bioanalysis. The magnetic processing module can introduce a magnetic field into parts of the cartridge, e.g., a processing chamber or other chamber or part of a chamber. This field can be used exert a magnetic force on magnetic and paramagnetic materials in the field, such as particles, such as beads, such as surfaces. Such particles can be sequestered from fluids in the chamber and, ultimately, separated from the fluids. In some embodiments the beads have a surface chemistry that facilitates the purification of the biologicals in conjunction with the chemical conditions. In other embodiments the bead have affinity molecules comprised of antibodies, aptamers, biomolecules, etc. that specifically purify certain biologicals such as cell types, organelles, nucleic acids, nuclei, or other components of tissue or samples.

200 120 1000 1050 1200 1250 200 500 In another aspect provided herein is a device for the dissociation and single-cell or single nuclei library preparation of a biological sample, the device comprising: (i) a chamber or area to input a biological sample or specimen; (ii) a cartridgecapable of dissociating tissue specimensinto single-cellsor single nucleiand then producing single-cell librariesor single-nuclei libraries; (iii) an instrument to operate the cartridgeand provide fluids as needed (iv) a measurement subsystemsuch as an optical imaging to measure titer, clumping, and/or viability, (v) exchange of dissociation solution for buffer at the desired titer, (vi) a magnetic processing or other processing chamber or tubing to perform magnetic separations, normalizations, purifications, and other magnetic processes, for example, to purify nucleic acids, couple enyzmatic reactions such as library preparation reactions, and other processes including producing single-cells or nuclei in isolation, such as nanodrops, nanoboluses, or physical separation or solutions including agarose, polyethylene gycol, and other chemicals and materials that slow diffusion, (vii) output vessels such as 8 well strip tubes, microtiter plates, Eppendorf tubes, a chamber in the cartridge, or other vessels capable of receiving cell suspensions including nanowells.

6001 6002 200 1000 200 6001 6002 In another embodiment, herein is a device and method for the dissociation of tissue into single cells which are then used to form microtissuesor organoidson the cartridgeor the single-cellsare used off the cartridgeto create microtissuesor organoids.

100 101 100 101 The basic elements of the Singulation Systemcan be configured in multiple ways depending on the specimen(s)and analytes to be analyzed. In the following examples, a few of the numerous configurations are described in detail but in no way is the invention limited to these configurations as will be obvious to one skilled in the art. The Singulation Systemcan accommodate many different types of specimens, comprised of fresh tissue; snap-frozen tissue; microtome slices (cryo, laser or vibrating) of tissue; fixed tissue; FFPE; bulk material obtained by surgical excision, biopsies, fine needle aspirates; samples from surfaces, and other matrices.

6001 6002 1000 120 1000 6001 6002 There is a need to fill gap in the single-cell sample preparation for microtissuesor organoidsby starting the workflow at processing raw solid tissues into single-cellsuspensions. The instant disclosure teaches how to produce a system that processes tissue specimensand other samples into single cellsuspensions and then form microtissuesor organoidswith little or no intervention by the operator once the process is started. This requires adapting to the widely varying starting types of tissue, with different requirements depending on the tissue, species, age, and state. In some embodiments, the cells are used to isolate tumor infiltrating lymphocytes which can be characterized by sequencing or flow cytometery, or cultured with lymphokines such as interleukin-1 to produce therapeutic tumor infiltrating lymphocytes. The therapeutic tumor infiltrating lymphocytes can then be infused into the patient to lyse tumor cells and combat disease progression.

2000 2200 2400 2500 3000 In the instant invention, many embodiments are possible and are incorporated by reference from patent application PCT/US2017/063811 filed Nov. 29, 2017 (Jovanovich, Chear, McIntosh, Pereira, and Zaugg, “Method and Apparatus for Processing Tissue Samples”) and from provisional patent application 62/679,466 filed Jun. 1, 2018 (Jovanovich, “Method and Apparatus for Processing Tissue Samples); the contents of all are incorporated herein in their entirety as well as the number system used therein. Systems with increasing capabilities can be developed as a series of embodiments, six are described: two embodiment as a Single Sample Singulator System, one as a Two Sample Singulator System, a Four Sample Singulator System, an Enhanced Singulator System, and the Single Librarianembodiments.

1000 6001 6002 100 100 This disclosure describes how to automate, integrate, and importantly standardize the complete process to create single-celland then produce microtissuesor organoidsin a single Singulator Systemsystem embodiment. The Singulator Systemwill greatly enable basic researchers, students, and translational researchers as well as clinicians and others with its ease of use and high performance.

Single-Use Cartridge Designs.

200 1000 1050 1000 6001 6002 Cartridgescan be used to process tissue into single-cellsuspensions or nucleiand are preferrably single-use. The major workflow steps to produce single-cell suspensionsfor the production of microtissuesor organoidsis to first mechanically disrupt solid tissue by enzymatic dissociation, and straining to remove clumps.

5 FIG. 19 20 FIGS.and 200 101 1000 6001 6002 6200 200 200 200 490 336 361 362 Referring to, cartridgewill input specimenand output viable singulated cellsthat are used to create microtissuesor organoidsor in some embodiments, as illustrated inby a hanging dropletin the cartridge. It is desirable that disposable cartridgeprocess multiple types of samples with mechanical disruption and enzymatic or chemical dissociation according to the tissue type and condition. The cartridgecan be designed to process tissue as quickly and as gently as possible, not expose the operator to the tissue being processed, and be manufacturable at low cost. Multiple mechanical methods may be needed to accommodate the wide range of tissues and their individual requirements: designs are shown that can be readily adapted to multiple different mechanical disruption methods comprising variable orifice, grinding with rotating plungers, pestles, and straining and filtering using a plungeras well as other mechanical methods without limitation.

200 200 2130 200 493 491 200 Cartridgescan be designed for 3D printing, injection molding in plastics with single or double pulls and low labor assembly, or layered assembly of fluidic and other layers, combinations of methods, and other methods well known to one skilled in the art. Fluids can be delivered to cartridgeby a syringe pumpor can be preloaded onto cartridgeor many combinations. In some embodiments, flexible tubingcan connect chambers and creates simple pinch valvesto direct flow or can be used to create a peristaltic pump. In other embodiments, channels are created in the cartridgeand valves can be incorporated such as pneumatic valves, or other valves.

Singulator System Embodiment

50 80 100 1000 1050 1100 1105 1072 1210 1000 1050 1072 1200 1250 1210 1060 1070 1071 1072 1073 1074 6001 6002 2 FIG. In one embodiment of the Sample Processing Systemas a Tissue Processing System, as shown in, the Singulator Systemcan perform powerful integrated tissue-to-genomics or sample-to-other answer (genomic, proteomic, metabolomic, or epigenetic, multi-omics, etc.) analysis functionality for scientists to simply and standardize the production and or analysis of single-cellor nucleisuspensions, affinity purified single cells, affinity purified nuclei, nucleic acids, and bulk librariesfrom solid or liquid tissues. As will be obvious to one skilled in the art, the biological materials produced such as single cells, nuclei, nucleic acids, single cell libraries, single nuclei libraries, bulk libraries, or other biocomponents comprised of subcellular components, or biomoleculessuch as macromoleculesand nucleic acids, comprised of DNAand RNA, or microtissues, or organoidscan also be used for many genomic, cell biology, proteomics, metabolomics, and other analytical methods.

100 500 The Singulator Systemcan integrate the preparation of biological materials from liquid or solid tissue(s) with measurement subsystemsthat perform an analysis selected from one or more of: DNA or RNA sequencing, next generation DNA or RNA sequencing, next next generation DNA or RNA sequencing of nucleic acids and their adducts such as epigenetic modifications; nanopore sequencing of nucleic acids and their adducts; single cell DNA sequencing of nucleic acids and their adducts; single nuclei RNA sequencing of nucleic acids and their adducts; PCR, digital droplet PCR, qPCR, RT-qPCR; genomic analysis, gene expression analysis, gene mapping, DNA fragment mapping; imaging including optical and mass spectrometry imaging; DNA or RNA microarray analysis; fluorescent, Raman, optical, mass spectrometery and other detection modalities of nucleic acids acids and their adducts with and without labels; proteomic analysis including fluorescent, Raman, optical, mass spectrometery, protein sequencing, and other detection modalities of proteins and peptides and their adducts and modifications with and without labels; carbohydrate characterization and profiling including sequencing, fluorescent, Raman, optical, mass spectrometery, and other detection modalities of carbohydrates and their adducts and other covalent polymers with and without labels; lipid characterization and profiling including sequencing, fluorescent, Raman, optical, mass spectrometery, and other detection modalities of lipids and their adducts and other covalent polymers with and without labels; flow cytometry; characterization of cells and profiling including fluorescent, Raman, optical, mass spectrometery, and other detection modalities of cells and their adducts and other covalent polymers with and without labels; metabolic profiling including sequencing, fluorescent, Raman, optical, mass spectrometery, and other detection modalities of metabolites and their adducts and other covalent polymers with and without labels; functional analysis including protein-protein interactions, protein-lipid interactions, protein-DNA interactions, RNA-DNA interactions, and other interactions between molecules derived from biological materials, with and without labels; bioinformatic analysis of cells, organelles, and biomolecules; and mass spectrometry and other analytical methods.

800 900 2000 2200 2400 200 440 1000 1200 1072 1070 1060 460 1100 1105 1072 1070 1060 440 460 450 468 430 In this preferred embodiment a Cell Singulation moduleand a Magnetic Processing moduleare integrated into a Single-Sample Singulator Systemor into a Two-Sample Singulator Systemor a Four-Sample Singulator Systemor other Singulator system that processes more than four samples. Mechanical and enzymatic dissociation is performed in single-use cartridgesin one or more preprocessing chambersto produce single-cell suspensionor nuclei suspensions, nucleic acids, biomolecules, subcellular components, or other products from pre-processing. The samples can then be processed in the one or more processing chamber(s)by optional bead-based affinity purification of cell types by surface antigens to produce affinity purified single-cell suspensionsor nuclear suspensions by nuclear antigensor nucleic acids, biomolecules, subcellular componentscan be further processed into purified mRNA, NGS libraries, or other sample types. In some embodiments, one or more of the preprocessingand processing chambersand strain chambersand vacuum trap chambersand waste chambersor other chambers can be combined.

2000 411 1430 2050 200 120 1000 1050 1000 1050 In a preferred embodiment, a Single-Sample Singulator Systemwas designed with reagentson-board or in a reagent moduleadjacent to the Single-Sample Singulator instrumentand with cartridgesincorporating one or more tissue-specific mechanical disruption modalities to accommodate the wide diversity of processing needs for tissue specimens. The system can input raw, unprocessed tissue samples and output single-cellsor nucleiin suspension, ready for processing into single cell NGS libraries off device or can process the single cellsor nucleiinto bulk libraries on the system or perform analysis of the processed tissues.

100 200 1000 800 2000 300 400 1000 1050 1000 1050 411 1430 1000 6001 6002 5 FIG. 6 FIG. The Singulator Systemcan mechanically disrupt tissue and enzymatically dissociate the disrupted tissue in a cartridgeinto single-cells. As shown in thein one embodiment, a Cell Singulation moduleor, as shown in, a Single Sample Singulator Systemcan combine the Physical Dissociation Subsystemand the Enyzmatic and Chemical Dissociation Subsystemto produce single-cellor nucleisuspensions. The instrument provides the mechanical motion and fluidics to the cartridge which in turn mechanically and enzymatically or chemically process the tissue into single cellsor nuclei. Multiple reagentscan be stored on the instrument or reagent modulewith cooling as needed. The single cellsuspension can in turn be used to generate microtissuesor organoids.

800 101 200 410 800 2110 2120 210 5 FIG. The Cell Singulation moduleas shown conceptually incombines the mechanical disruption of specimenon cartridge, adds enzymatic or chemical dissolution solutionand other fluids according to the protocols, and controls sample movement, pressures, and temperature. The Cell Singulation modulecan move or rotate mechanical tissue disruptor elements comprised of without limitation a syringe plunger, pestle, Dounce pestle, or grinder, using a z axis stepperwith a rotary motorcoupled through the cap.

2000 2110 2120 725 6 FIG. 7 8 9 10 FIGS.,,, and A 3D CAD representation of one embodiment of a Single-Sample Singulator Systemdesign packaged with a ‘skin’ is shown inand another embodiment is shown in. Both embodiments have a two axis mechanical motion (Z axis stepperand rotary motor) integrated with fluidics based on a syringe pump, for example, with 1.6 μL resolution with a six-way valve (C2400MP, TriContinent) controlled by control software.

6 FIG. 720 725 730 750 1010 Referring to, a computerwith an operating system, for example, such as Windows 10 and 85 Gbytes HD (Beelink, AP42), can run control softwareto control the system with display on a 10″ touchscreen(eleduino, Raspberry Pi10) or on a tablet. Chassisprovides the framework to mount components and the exterior case of the system.

2000 600 2130 2140 200 415 200 440 460 900 200 1000 1050 6001 6002 1072 1070 1060 6 FIG. The embodiment of the Single-Sample Singulator Systemshown inhas a fluidic subsystemwith a single syringe pumpwith a single six-way valveto supply liquids, pressure, or vacuum to cartridgefrom reagent block. In one embodiment, cartridgehas two preprocessing chambersand a single processing chamber. In a preferred embodiment, magnetic processing modulecan apply magnetic force to cartridgeunder software control to enable the use of paramagnetic beads, paramagnetic surfaces, paramagnetic nanoparticles, and other magnetic or paramagnetic particles to purify and analyze single cells, including stem and other types of cells, nuclei, microtissues, organoids, nucleic acids, biomolecules, subcellular components, or other products.

2000 1430 2050 2050 1430 7 FIG. 7 FIG. 6 FIG. A preferred embodiment of the Single-Sample Singulator Systemwith a case on is shown in. This embodiment has a reagent modulewhich can be separate from Single Sample Singulator Instrumentas shown inwith power and control provided by Single Sample Singulator Instrumentor a separate power source and processor can be used or as shown inreagent modulebe integrated inside a single instrument case.

8 FIG.A 8 FIG.A 1430 1420 1421 1420 720 725 1421 1419 1417 1420 411 1419 1418 411 As shown in, in a preferred embodiment reagent modulehas reagent Peltierattached to temperature distribution plate. The temperature of reagent Peltiercan be changed under control of computerand control softwareto heat or cool temperature distribution plateinside reagent storage chamberby monitoring temperature sensor, which may be a thermocouple, or a thermistor, or optical detection of a thermochromic surface or other method. In a preferred embodiment, as shown in, reagent Peltiermaintains a set of reagentsat 4° C. in temperature-controlled reagent storage chamberand room temperature reagent storage chambermaintains a second set of reagentsat ambient temperature. It will be obvious to one skilled in the art that embodiments can have a one or more temperature controlled chambers containing one or more reagents.

8 FIG.B 1419 1422 1423 1424 2130 1430 1425 2240 2050 1420 1430 1420 1430 2050 1424 1426 1427 1428 1426 1428 1428 411 1427 1430 1417 Referring to, reagent storage chamberhas insulationand lid. Fluidic bundlefluidically connects syringe pumpwith reagent module. In one embodiment, a power and control bundlefrom reagent Peltier relay boardon Single Sample Singulator Instrumentcontrols reagent Peltier. In another embodiment, reagent moduleis powered by separately plugging into electrical power and reagent Peltieris controlled by a separate microprocessor, allowing reagent moduleto operate independently of Single Sample Singulator Instrumentand is connected fluidically by fluidic bundlecomprised of tubing such as 1/16 ID tygon tubing or other tubing, capillaries, microchip, or other fluidic vessels. In some embodiments, reagent containerhas reagent container lidcontains one or more reagent container sensorsto monitor the amount of reagent in the container, for example by weight, or by an phase interface using optics or other electromagnetic measurement methods, or by conductivity, or to determine the identity of reagent containerby RFID, EEPROM, or other identification technologies. Information from reagent container sensor(s)can be stored in system log or be used to alert users to issues with reagent container sensoror other actions such as the need to changes reagents. In some embodiments, reagent container lidhas one or more openings that may allow tubing or capillaries or fittings to be inserted or a hole with an optional filter. In a preferred embodiment, reagent modulehas reagent Peltier exhaust duct.

9 FIG. 2050 2110 2120 2111 1020 1010 2110 101 2130 2141 2142 200 1430 Referring to, in a preferred embodiment, Single Sample Singulator Instrumenthas z-axis stepper motor, which may have an optional encoder, that controls the vertical position of rotary motormounted on z-axis stepper slideattached to the inverted ‘U’ shaped structural framemounted on chassis. A force gauge can be incorporated into the z-stage stepperto provide force-feedback control of the mechanical force on the specimen; this can help ensure very gentle mechanical processing steps. Syringe pumpconnects fluidically with tubing or capillaries or microchips or other fluidic connectors with six-way valveand six-way valveto supply reagents, pressure, or vacuum to cartridge(not shown) from reagent module.

200 1510 1450 200 440 2121 2120 200 1510 1450 1480 1452 1450 200 1470 1415 1500 Cartridgeis placed into cartridge receiver trayon cartridge slidewhich is designed to hold cartridgeprecisely, with the center of preprocessing chamberconcentric with the center of rotary motor shaftof rotary motorwithin a distance or 1 or, 5, or 10, or 15, or 20, or 25, or 50, or 100, or 250 μm, or more when inserted by moving cartridgein cartridge receiver trayon cartridge slideon cartridge slide railuntil spring-loaded cartridge slide knoblocks into place into a hole in cartridge slidewith cartridgeheld in place near or in contact with the thermal transfer plateand making fluidic connections with the pogo pinsof cartridge interface.

1475 1470 1440 440 460 2250 440 460 The temperature regulating subsystemcan set the thermal transfer plateto a given temperature by cartridge Peltieror other temperature regulating device such as strip resistive heaters, circulating fluids, etc. to set the cartridge temperature in the preprocessing chamberand processing chamberunder control of board. In some embodiments, the temperature of the preprocessing chamberand processing chambercan be set independently.

200 1415 200 1415 1416 200 1416 2130 1470 200 1416 In a preferred embodiment, fluidic ports on cartridgedock with spring-loaded pogo pinsto connect fluids, gases, or vacuum to cartridgeon cartridge insertion. In another embodiment, pogo pinsor canulaare moved to connect with cartridgeafter cartridge insertion. In another embodiment, canulaconnected to fluidic lines from syringe pumpare held rigidly attached to the thermal transfer plateor other part of instrument and cartridgehas flexible materials on cartridge ports that seal with the canula(s)after cartridge insertion, as described below. Cartridge ports are ports opening out of a cartridge. A cartridge port may communicate directly with a chamber by being a port in the chamber, or indirectly. e.g., through another chamber comprising the port and communicating with the chamber in question.

2000 900 910 935 1020 725 2122 910 200 685 200 910 200 685 9 FIG. 9 FIG. The embodiment of the single-sample Singulator Systemshown inhas a Magnetic Processing Moduleand magnetis moved by magnetic actuatormounted on inverted ‘U’ shaped structural frameunder control of control softwareusing controller. Magnetcan be far from cartridgeas shown inand not interact with any magnetic beadsin cartridgeor in an extended position magnetis moved to be near cartridgefor magnetic capture and processing of magnetic beads.

10 FIG. 2000 1021 1020 710 2122 2112 2230 2225 2000 762 761 760 2141 2142 2210 2212 2240 1420 Referring to, in a preferred embodiment, the Single-Sample Singulator Systemhas a back structural frameon structural framethat mounts electronicscomprising rotary motor controller, z-axis stepper controller, 24 V to 5 V step down power supplyand 24 V to 12 V step down power supply. Power can be supplied to single-sample Singulator Systemby plugging a 24 V power supply into plugconnecting to fuseand power switch. Six way valvesandare controlled by boardsand. Reagent Peltier relay boardcan control reagent Peltier.

11 FIG. 11 FIG. 2200 101 200 2110 2120 2111 1025 1010 2130 2141 2142 200 1430 1415 440 460 1500 Singulator systems that process one or more cartridges simultaneously are within the scope of the present invention.illustrates a Two Sample Singulator instrumentthat can process two specimensin two cartridges. The embodiment shown inhas two z-axis stepper motorsthat independently controls the vertical position of two rotary motorsmounted on two z-axis stepper slidesattached to the inverted ‘M’ shaped structural framemounted on chassis. Syringe pumpconnects fluidically with tubing or capillaries or microchips or other fluidic connectors with six-way valveand six-way valveto supply liquids, pressure, or vacuum to cartridgesfrom reagent module(not shown) through pogo pins(not shown) mounted above thermal transfer plate preprocessing chamberand processing chamber. A third 6 way valve (not shown) can provide fluids to the second cartridge interface.

200 440 460 435 468 The cartridgecan have one or more Pre-Processing Chamber(s)and none, one, or more Processing Chamber(s)as well as none, one or more other chambers such as cartridge waste chamberor vacuum trap chamber.

12 13 FIGS.and 210 440 101 120 425 440 200 200 1415 1416 470 440 485 460 467 468 In a preferred embodiment, illustrated in, cap, alternatively referred to as a tissue disruptor, is placed on top of preprocessing chamberafter specimenor tissue specimenis added through sample inlet portinto preprocessing chamberof cartridge. After cartridgeis inserted into the instrument, pogo pins, canula, or other fluidic connectors can connect with none, one, or more of cartridge portsto supply reagents to preprocessing chamber, cartridge portto supply reagents or vacuum to processing chamber, and cartridge vacuum trap portto supply vacuum to vacuum trap chamber.

12 FIG. 440 460 453 471 452 2711 460 450 2711 347 440 453 462 462 460 468 465 462 201 2711 200 A preferred embodiment illustrated influidically connects preprocessing chamberto processing chamberusing fluidic line, which can be tubing, connecting from preprocessing chamber nippleto lid nipplepositioned over strainerinserted into processing chamber, eliminating the need for a separate strain chamber. In other embodiments, strainercan be incorporated as an in-line filter, for example in a swinney filter holderattached to the output of preprocessing chamberor in fluidic lineor attached to lid. Lidproduces a vacuum tight seal of processing chamberand vacuum trap chamberwhen capis closed onto lid, and can be attached to cartridge bodyby ultrasonic welding, glue, epoxy, adhesives, and other methods to produce a vacuum tight seal and prevents changing strainerensuring single usage of cartridge.

200 491 453 520 453 2160 200 2160 2150 200 200 In some embodiments, cartridgecan have on-cartridge valves which can be pinch valveson fluidic lines such as fluidic linewhich the instrument actuates to open and close lines, or by using a ‘T’ junction and two lines, rout fluids down different paths such as to a optics imaging system. In another embodiment, fluidic lines such as fluidic linecan be partially closed to create a variable orificethat can disrupt partially dissociated tissue. Actuators can open and pinch close tubing in the cartridge, or operate the variable orificeusing variable orifice devicewhen desired. In other embodiments, cartridgecan have on-cartridge valves which can be miniaturized pneumatic valves, or microvalves. In some embodiments, microfluidics or microchips are used for fluidic lines. In a preferred embodiment there are no valves on the cartridgewith all fluidic control from the instrument.

13 FIG.A 467 485 1000 1050 1060 1070 440 453 2711 451 461 460 2711 2711 110 210 211 218 425 218 355 6001 6002 Referring to, when vacuum is applied to vacuum trap portor to reagent port, liquids including single cell suspensions, nuclei, and other subcellular components, and biomoleculesare pulled from preprocessing chamberthrough fluidic lineand strainerinto strain drainand into output collector regionof processing chamber. Strainercan have pore sizes such as 2, 5, 10, 15, 20, 25, 30, 40, 50, 70, 100, 125, 200 μm, or larger to filter the suspension of biological material. Multiple in-line or stacked strainerscan be employed to successively remove different sized components of the dissociated tissue specimen. Capwith cap coupler, and headis shown ready to be inserted into sample inlet port. Headcan have a surface for disrupting tissue that can comprise raised featuresthat aid in mechanically disrupting a tissue, organ, microtissue, organoidor other biological material.

13 FIG.B 13 FIG.C 211 212 214 211 216 214 218 353 355 211 216 218 336 345 Referring toand, the cap coupler (also referred to as “drive head”)is held inside cap sheathwhich in one embodiment has cap sheath hole. Cap coupleris attached to cap shaftwhich passes through cap sheath holeand is attached to the headwhich can be a rotorwith grinding teeth. The assembly of cap couplerattached to cap shaftand headare referred to as a plungerwhich is a type of moveable mechanical tissue disruptor.

14 FIG.A 14 FIG.B 218 216 356 218 440 221 2110 211 2125 218 356 357 440 218 218 221 218 440 211 440 357 356 357 222 218 357 357 221 218 345 222 218 345 440 440 221 222 Referring to, in a preferred embodiment, headattached to cap shafthas a outwardly annular beveled head featuredesigned to improve centricity of headinside preprocessing chamberand thereby the uniformity of side gapat the bottom of travel. When z-axis stepper motorlowers and cap coupleris pushed down by rotary motor coupler, headwill lower until outwardly annular beveled featureengages with inwardly annular beveled preprocessor chamber featureon the inside wall of preprocessing chamberto produce a centered headas shown in. The centering of headwill produce a uniform side gapbetween headand the inner wall of preprocessing chamber. In addition, if the height of headis less than the height of the preprocessing chamberbelow inwardly beveled feature, the engagement of outwardly annular beveled head featurewith inwardly annular beveled preprocessor chamber featurewill set a uniform bottom gap. The size of the side gap and the bottom gap can be optimized for different cell types or for different sized nuclei or subcellular organelles, or multicellular structures such as intestinal crypts. In addition, to allow passage of disrupted tissue when headis seated on inwardly annular beveled preprocessor chamber feature, the inwardly annular beveled preprocessor chamber featurecan be fluted to have sections with the same or different depths. The side gapbetween the headof moveable mechanical disruptorand the inside wall is preferrably greater than or equal to 1 μm, or 2 μm, or 5 μm, or 10 μm, or 15 μm, or 20 μm, or 25 μm, or 30 μm, or 40 μm, or 50 μm, or 75 μm, or 100 μm, or 150 μm, or 200 μm, or 250 μm, or 500 μm, and 1000 μm or more, as well as any size in between. The bottom gapbetween the bottom of headof moveable mechanical disruptorand the bottom of preprocessing chamberis preferrably greater than or equal to 1 μm, or 2 μm, or 5 μm, or 10 μm, or 15 μm, or 20 μm, or 25 μm, or 30 μm, or 40 μm, or 50 μm, or 75 μm, or 100 μm, or 150 μm, or 200 μm, or 250 μm, or 500 μm, and 1000 μm or more, as well as any size in between. In some embodiments, different heads can be selected to be used with the same diameter preprocessing chamberto produce different side gapsor bottom gapsto simplify manufacturing and inventory management requirements. A bottom gap between a flat surface of the head and the flat bottom surface of the preprocessing chamber can also be limited by the position of the flutes, or half domes, or other structures that prevent or define gaps between a flat surface of the head and the flat bottom surface of the preprocessing chamber.

15 FIG. 15 FIG.A 15 FIG.B 15 FIG.C 15 FIG.D 200 442 442 441 442 470 1416 1415 446 442 442 1416 1415 1416 1415 1415 1416 442 443 442 442 442 442 444 442 Referring to, none, one, or more of the ports to cartridgecan have flexible or low durometer port covers, for example without limitation 40 to 100 durometer. As illustrated inand in cutout, port covercan be inserted into the space between the port and port cover retaining cylinderto secure the port coverin place over, for example as shown, reagent addition port. A fluidic canulaor fluidic pogo pinwith an outside diameter larger than port cover center holecan engage the port covered by port coverand, because of the relatively low durometer, the port coverwill be deformed by fluidic canulaor fluidic pogo pinto create a seal around the fluidic canulaor fluidic pogo pin. In some configurations, the deformation can be used to eliminate the need for springs and the use of the fluidic pogo pincan be replaced by a non-movable fluidic canula.shows port coverretained by crimp seal.shows port coverretained by forming port cover retaining cylinderhigher than the port coverand melting the port cover retaining cylinderto form a heat stake lipthat retains port cover.

2050 2110 2120 2121 2125 211 210 200 2125 211 2125 211 210 2110 2125 2121 211 210 2125 217 2125 217 211 217 211 217 2121 2125 217 211 2110 2125 217 2110 2120 2121 2125 217 211 213 212 218 218 440 354 218 2110 2120 2125 213 211 2125 16 FIG. 16 FIG. 16 FIG.C 16 FIG.D 16 FIG.E In a preferred embodiment the Single Sample Singulator Instrumenthas an actuator for mechanical processing that has a stepper motorthat controls the vertical position of rotary motorand rotary motor shaftattached to rotary motor couplerthat in turn can mechanically couples with cap couplerof the capwhen inserted into cartridge. The coupler can have a drive head that takes any appropriate form, such as a slot, a phillips head, a quadrex, atri-wing, aspanner or a hex. Rotary motor couplerhas one or more facets that reversibly engage cap couplerby actions such as moving downward and slowly rotating. As shown inA, in a preferred embodiment, rotary motor couplerhas a single blade to engage cap couplerin cap. As shown in the cutaway inB, when stepper motorlowers, the rotary motor couplerattached to rotary motor shaftengages cap couplerin capand if the rotary motor coupleris not lined up with cap coupler groove, the rotary motor couplercan not directly insert into the cap coupler groove. In a preferred embodiment, cap couplerhas two surfaces on either side of cap coupler groovewhich slope in opposite directions across the cap couplersuch that each side has a higher and lower wall on either side of cap coupler groove. When rotary motor shaftturns in the clockside direction (looking from above), rotary motor couplerblade spins in the clockside direction and encounters the high side of the wall of cap coupler grooveand begin to rotate cap couplerclockwise. As stepper motorlowers, the rotary motor couplerwill engage the cap coupler groove, as shown in. As shown in, when stepper motorcontinues to lowers, the rotary motorand rotary motor shaftattached to rotary motor couplerwill lower, pushing on cap coupler grooveand the cap couplerwill compress cap springagainst the bottom of cap sheathand lower head. As shown in, headcan be lowered close to or in contact with the bottom of preprocessing chamber, which can be a stator, and headcan be rotated to disrupt tissue. When stepper motorraises, rotary motorand rotary motor couplerraise up and cap springdecompresses to push cap coupleragainst rotary motor couplerto continue engagement.

2050 2110 2120 345 210 211 345 218 In another embodiment of the Single Sample Singulator Instrument, stepper motorcontrols the vertical position of rotary motorwhich is magnetically coupled to moveable disruptorwith a magnetic or paramagnetic element embedded with capas part of cap coupleror as part of moveable disruptoror head.

2125 211 2120 325 218 2110 2120 2125 345 218 440 2120 2110 345 218 When rotary motor coupleris engaged with cap couplerby mechanical coupling, magnetic coupling, pneumatic, or fluidic coupling, or other coupling methods, and rotary motorrotates, moveable disruptorand headare rotated. Stepper motorcontrols the vertical position of the rotary motorand thereby the the vertical position of rotary motor coupler, to raise or lower moveable disruptorand headin preprocessing chamber. Combining rotation of rotary motorand movement of stepper motorenables many patterns of motion of moveable tissue disruptorand head.

440 440 218 345 The inside walls of preprocessing chambercan be embodied in many different shapes. The inside walls of preprocessing chambercan be fluted to have sections with different depths. In a preferred embodiment, the inside wall can have a circular profile with the largest gap between the headof moveable mechanical tissue disruptorand the inside wall of preferrably greater than or equal to 1 μm, or 2 μm, or 5 μm, or 10 μm, or 15 μm, or 20 μm, or 25 μm, or 30 μm, or 40 μm, or 50 μm, or 75 μm, or 100 μm, or 150 μm, or 200 μm, or 250 μm, or 500 μm, and 1000 μm or more, as well as any size in between.

345 345 218 353 355 353 355 354 440 120 1000 1050 1070 218 221 218 218 345 101 218 440 345 345 440 13 14 FIGS.and Moveable tissue disruptorcan be embodied in many different shapes with many different profiles. In one embodiment, moveable tissue disruptorcan have a headwhich is a rotorwith optional features, for example, grinding teethon the bottom of rotorand grinding teethon statorwhich is on the top surface of the bottom of the preprocessing chamberto assist in disruption of large pieces of tissue specimensinto smaller pieces or assist in the dissociation into single cellsor nucleior biomolecules. As shown in, the sides of headcan be a cylinder to create an inside gapwith the inside wall over the length of the cylinder. By raising and lowering headwithout turning head, thereby using it as a moveable disruptor, the system can process specimenby trituration. In another embodiment the sides of the headcan form a ball-like structure to create a gap with the inside wall in a small area and the bottom of preprocessing chambercan be rounded to match the ball-like structure to create a Dounce-like mechanical tissue disruptor. In other embodiments, multiple regions with gaps of the same or different sizes can be created by varying the side profile of moveable tissue disruptorand the inner wall of preprocessing chamber.

1000 1050 1000 1050 Disruption of tissue can include a plurality of disruption steps, each involving positioning the head a different distance from floor of the chamber to produce gaps of different sizes. Typically, at each position, the head will rotate, further facilitating disruption or mixing. In certain embodiments, an organ can be auto-minced by the disruptor before tissue disruption into single cellsor nucleior other biological materials. Such a method can involve a first disruption step, which can include setting the head at a plurality of different distances from the floor of the chamber and rotating at each gap distance, to provide tissue with greater surface area and less distance for access by enzymes. A next step can involve incubating the auto-minced organ with enzymes or chemicals for tissue disruption into single cellsor nuclei. A next step can involve a second disruption step, which, in turn, can include setting the head at a plurality of different distances from the floor of the chamber and rotating the head.

2000 101 1000 1430 210 200 425 210 440 212 440 1510 2050 1450 740 750 2050 440 101 13 FIG.A The Single Sample Singulator Systemcan be operated in many configurations. As an example, an operator may wish to process a fresh mouse kidney specimeninto a single cell suspensionand use reagents stored on Reagent Module. The operator would remove capfrom cartridgeas shown inand add a whole mouse kidney, or a part of mouse kidney, or part of a kidney that had been preminced to sample inlet port. The cap, which is a moveable disruptor, is replaced on preprocessing chamberwith the bottom of cap sheathseated on an annular seat in preprocessing chamber. The now complete cartridge with a tissue specimen is placed on cartridge receiver trayand inserted into the Single Sample Singulator instrumentwith cartridge slide. After the appropriate protocol is selected through user interfaceon tablet, the Single Sample Singulator instrumentheats thermal transfer plate to hold the preprocessing chamberat 37° C. and then begins processing kidney specimen.

2110 2120 2125 211 725 1419 1430 470 440 17 FIG. After initialization of electronic boards, the z-axis stepper motormoves the rotary motordown to engage rotary motor couplerwith cap coupler. The control softwarethen selects the proper valve settings to pull two mL of mouse kidney reagent solution from Position 3 in temperature-controlled reagent storage chamberof reagent module, as shown in, and deliver it through portto preprocessing chamberwhere the mouse kidney has been placed.

2110 2120 218 353 440 353 353 353 If selected by the protocol, an auto-mince procedure to macerate the tissue is performed by the z-axis stepper motormoving rotary motor, and therefore the mechanical tissue disruptor and head, which is functioning as a rotor, to 1.5 mm from the bottom of the preprocessing chamberand then rotoris rotated clockwise for four seconds and then counterclockwise for four seconds at 95 rpm. Rotoris lowered to 0.6 mm from the bottom and rotated clockwise for four seconds and counterclockwise for four seconds at 95 rpm. Rotoris lowered to 0.3 mm from the bottom and rotated clockwise for four seconds and counterclockwise for four seconds at 95 rpm to complete the standard automince portion of the protocol.

101 353 101 440 210 353 For mouse kidney, the now auto-minced kidney specimenis then incubated for twenty minutes with continuous top immersion mixing where rotoris lowered into the top third of the mouse kidney reagent solution with kidney specimenin preprocessor chamberand the rotary motorspins rotorclockwise at 95 rpm in a continuous immersion mixing mode while the enzymatic formulation digests the extracellular matrix in the solid tissue to release cells.

353 101 353 353 353 353 353 353 353 440 After 20 min, the tissue is mechanically disrupted by lowering rotoruntil it is 4.2 mm from the bottom, approximately 20% immersed into mouse kidney reagent solution with kidney specimen, and then the first mechanical disruption cycle is performed with rotorrotating clockwise for four seconds and then rotating counterclockwise for four seconds at 95 rpm. The second disruption cycle is performed by lowering rotorby 1.5 mm and rotating clockwise for four seconds and then counterclockwise for four seconds at 95 rpm. The third disruption cycle is by lowering rotorby 0.9 mm and and rotating clockwise for four seconds and counterclockwise for four seconds at 95 rpm. Then, the fourth and fifth disruptions cycles are performed with lowering rotorby 0.6 mm each cycle with rotation clockwise for four seconds, counterclockwise for four seconds, then rotation clockwise for four seconds, and counterclockwise for four seconds at 95 rpm for each disruption cycle. For the sixth disruption cycle, the rotoris raised 0.3 mm and then rotated clockwise for four seconds, counterclockwise for four seconds, clockwise for four seconds, and counterclockwise for four seconds at 95 rpm. For the seventh disruption cycle, the rotoris lowered 0.6 mm and rotated clockwise for four seconds, counterclockwise for four seconds, rotated clockwise for four seconds, and counterclockwise for four seconds at 95 rpm. For the eight and final disruption cycle, the rotoris lowered 0.3 mm in contact with the bottom surface of preprocessing chamberand rotated clockwise for four seconds, counterclockwise for four seconds, rotated clockwise for four seconds, and counterclockwise for four seconds at 95 rpm. Many other possible disruption profiles are enabled by this instant invention.

353 355 354 355 222 353 440 The mechanical tissue disruption occurs at two places: first, at the bottom of rotorby grinding teethand the top of statorwith complementary grinding teethto mechanically dissociate the solid tissue in bottom gapand secondly, the gap between the circumference of the rotorand the inner wall of preprocessing chamberacts as an orifice to disrupt the tissue.

353 440 2130 467 453 2711 451 461 460 With the rotorpositioned at the bottom of preprocessing chamber, syringe pumpthen pulls vacuum through the appropriate six way valve settings on vacuum trap portto pull the dissociated mouse kidney single cell suspension through line, through 70 μm strainerwhere it drains down strain drainand into output collector regionin processing chamber.

725 1418 1430 470 440 353 353 440 2130 467 453 2711 451 461 460 440 460 1000 465 461 17 FIG. The control softwaresets the selection of valve settings to pull two mL of HBSS-Ca-Mg from Position 13 in room temperature reagent storage chamberof reagent moduleas shown inand deliver it through portto preprocessing chamber. Rotorcan be moved to mix any remaining dissociated cells with the HBSS-Ca-Mg and then with rotorpositioned at the bottom of preprocessing chamber, syringe pumpthen pulls vacuum through the appropriate six way valve settings on vacuum trap portto pull the HBSS-Ca-Mg and any remaining dissociated mouse kidney single cells suspension through line, through 70 μm strainer, down strain drainand into output collector regionin processing chamber. This process is then repeated to deliver and pull a second two mL of HBSS-Ca-Mg through preprocessing chamberand into processing chamber. The mouse kidney single cellsuspension can then be pipetted out by opening processing chamber capand withdrawing the cell suspension from output collector regionusing a pipettor.

1000 1000 18 FIG.A The mouse kidney single cellsuspension can be centrifuged at 300 g for five min to collect the cells as a pellet, the red blood cells lyzed for five min with a RBC lysis buffer, and the suspension centrifuged at 300 g for five min to collect the cells. As an example, a 262 mg of mouse kidney produced a single cell suspensionby this process with a cell titer of 14,670,000 cells at a 85.5% viability as determined by counting on a Countess II with Trypan blue staining as shown in.

2000 218 218 410 101 218 218 Other tissues or organs may benefit from different modes of mixing. The Single Sample Singulator Systemis designed to perform a plurality of mixing modalities. For example, top mixing is designed to position the bottom of headat 15 mm above the bottom and rotate headto mix the enzymatic or chemical dissolution solutionwith the specimen. Shallow immersive mixing can be performed by continuously rotating headas it is moved from 17.7 mm above the bottom down to 16.8 mm and back up again. Tritutation mixing can be performed by moving headwithout rotation from 12.3 mm above the bottom down to 0.3 mm above the bottom. Many other mixing modalities are enabled.

2000 1050 101 1050 1430 210 200 425 210 440 101 1510 2050 1450 740 750 2050 1470 440 460 101 1470 13 FIG. The Single Sample Singulator Systemcan be operated in many configurations to produce nucleisuspensions. As an example, an operator may wish to process a fresh mouse kidney specimeninto a single nuclei suspensionand use reagents stored on Reagent Module. The operator would remove capfrom cartridgeas shown inand add a whole mouse kidney, or a part of a kidney, or part of a kidney that had been preminced to sample inlet port. The cap, which is a tissue disruptor, is replaced on preprocessing chamberand the now complete cartridge with a tissue specimenis placed on cartridge receiver trayand inserted into the Single Sample Singulator instrumentwith cartridge slide. After the appropriate protocol is selected through user interfaceon tablet, the Single Sample Singulator instrumentcools thermal transfer plateto hold the preprocessing chamberand processing chamberat 4° C. and then begins processing kidney specimen. The thermal transfer platecan also be preheated or precooled as needed.

2110 210 2125 211 725 412 1419 1430 470 440 17 FIG. After initialization of boards, the z-axis stepper motormoves the rotary motordown to engage rotary motor couplerwith cap coupler. The control softwarethen selects the valve settings to pull two mL of nuclei isolation solutionfrom Position 1 in temperature-controlled reagent storage chamberof reagent moduleas shown inand deliver it through portto preprocessing chamber.

218 353 412 101 345 218 353 353 353 353 353 353 353 The tissue is then mechanically disrupted by lowering headwhich will function as rotoruntil it is 4.2 mm from the bottom, approximately 20% immersed into the nuclei isolation solutionwith kidney specimen, and then the first mechanical disruption cycle is performed with moveable mechanical disruptorand headacting as a rotorrotated clockwise for four seconds and then rotated counterclockwise for four seconds at 135 rpm. The second disruption cycle is by lowering rotorby 1.5 mm and rotating clockwise for four seconds and then counterclockwise for four seconds at 135 rpm. The third disruption cycle is by lowering rotorby 0.9 mm and and rotating clockwise for four seconds and then rotating counterclockwise for four seconds at 135 rpm. Then, the fourth and fifth disruptions cycles are performed with lowering rotorby 0.6 mm with rotation clockwise for four seconds, counterclockwise for four seconds, rotation clockwise for four seconds, and counterclockwise for four seconds at 135 rpm for each disruption cycle. For the sixth disruption cycle, the rotoris raised 0.3 mm and then rotated clockwise for four seconds, counterclockwise for four seconds, clockwise for four seconds, and counterclockwise for four seconds at 135 rpm. For the seventh disruption cycle, the rotoris lowered 0.6 mm and rotated clockwise for four seconds, counterclockwise for four seconds, rotated clockwise for four seconds, and counterclockwise for four seconds at 135 rpm. For the eighth disruption cycle, the rotoris lowered 0.3 mm and rotated clockwise for four seconds, counterclockwise for four seconds, rotated clockwise for four seconds, and counterclockwise for four seconds at 135 rpm.

353 355 354 355 222 353 440 221 The mechanical tissue disruption again occurs both at the bottom of rotorby grinding teethand the top of statorwith complementary grinding teethmechanically dissociating the solid tissue in bottom gapas well as any tissue passing between the circumference of the rotorand the inner wall of preprocessing chamberin side gap.

353 440 2130 467 453 2711 460 451 461 With the rotorpositioned at the bottom of preprocessing chamber, syringe pumpthen pulls vacuum through the appropriate six way valve settings on vacuum trap portto pull the dissociated mouse kidney nuclei suspension through line, through a 40 μm strainerin processing chamber, down strain drainand into output collector region.

725 413 1419 1430 470 440 353 1050 413 353 440 2130 467 413 1050 453 2711 451 461 1050 465 461 17 FIG. The control softwaresets the selection of valve settings to pull two mL of nuclei storage solutionfrom Position 2 in temperature-controlled reagent storage chamberof reagent moduleas shown inand delivers it through portto preprocessing chamber. Rotorcan be moved to mix any remaining dissociated nucleiwith the nuclei storage solutionand then with rotorpositioned at the bottom of preprocessing chamber, syringe pumppulls vacuum through the appropriate six way valve settings on vacuum trap portto pull the nuclei storage solutionand any remaining dissociated mouse kidney single nucleisuspension through line, through a 40 μm strainer, down strain drainand into output collector region. The mouse kidney single nucleisuspension can then be pipetted out by opening processing chamber capand withdrawing the cell suspension from output collector region.

1050 413 1050 1050 18 FIG.B The mouse kidney single cellsuspension can be centrifuged at 500 g for 5 min to collect the cells as a pellet before resuspension in nuclei storage solutionor other media. As an example, a 108 mg mouse kidney produced by this process yielded a nuclei suspensionwith a titer of 24,225,000 as determined by counting on a Countess II with Trypan blue staining, a picture of the nuclei suspensionis shown in.

FFPE tissue is commonly used by pathologists to examine biopsy samples. Massive banks of FFPE tissue contain archives of tissue samples from many disease states including cancers. Currently, isolating single cells or nuclei from FFPE is challenging and not automated.

200 210 200 2050 200 471 471 In one embodiment, one or more thin sections from an FFPE block are added into cartridge, the capadded, and the cartridgeplaced into the Single Sample Singulator instrument. In some embodiments cartridgehas a filter, such as a 25 μm filter added in or over the channel leading to preprocessing chamber nippleto prevent loss of the undissociated FFPE thin section through the preprocessing chamber nipple.

1430 1430 460 460 491 460 After selection of the appropriate cell or nuclei FFPE protocol, and using the appropriate setup of reagent module, the instrument can add, for example, 2 mL of xylol from the reagent moduleand incubate for a time period selected from the range of 10 sec, 30 sec, 1 min, 5 min, 10 min, 15 min, 30 min or longer at room temperature or other temperature. The xylol is then pulled into the processing chamberas described and the process repeated two additional times with xylol. Xylene, histolene, and other compatible solvents can replace xylol. In some embodiments, the volume of processing chamberis enlarged to accommodate the deparafinnization materials. In other embodiments, a separate waste chamber is added and pinch valvesare used to direct flow either to a waste chamber or processing chamber.

1430 200 460 The instrument can then perform reverse sequential ethanol washes, for example, by adding two mL of 100% ethanol from the reagent moduleto cartridgeand incubating for a time period selected from the range of 10 sec, 30 sec, 1 min, 5 min, 10 min, 15 min, 30 min or longer at room temperature or other temperature. The 100% ethanol is then pulled into the processing chamberas described and the process repeated none, one, or more additional times with 100% ethanol.

1430 200 460 The instrument can add 2 mL of 70% ethanol from the reagent moduleto cartridgeand incubate for a time period selected from the range of 10 sec, 30 sec, 1 min, 5 min, 10 min, 15 min, 30 min or longer at room temperature or other temperature. The 70% ethanol is then pulled into the processing chamberas described and the process repeated none, one, or more additional times with 70% ethanol.

1430 200 460 The instrument can add 2 mL of 50% ethanol from the reagent moduleto cartridgeand incubate for a time period selected from the range of 10 sec, 30 sec, 1 min, 5 min, 10 min, 15 min, 30 min or longer at room temperature or other temperature. The 50% ethanol is then pulled into the processing chamberas described and the process repeated none, one, or more additional times with 50% ethanol. In some embodiments, a 30% ethanol step or other additional reverse sequential ethanol wash steps can be added. In some embodiments, the ethanol washes and other solutions can be supplemented with PBS, bovine serum albumin, RNAse inhibitors, protease inhibitors, or other supplements.

200 460 460 The instrument can add 2 mL of purified water, such as double distilled water with RNAse inhibitors, from the reagent module to cartridgeand incubated for a time period selected from the range of 10 sec, 30 sec, 1 min, 5 min, 10 min, 15 min, 30 min, 60 min, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours at at 4° C., room temperature or other temperatures. The water is then pulled into the processing chamberas described and the process repeated none, one, or more additional times with purified water. The waste in processing chambercan be removed at this time or previously as needed if it has not been directed to a waste chamber.

The deparaffinized FFPE can then be treated by different methods. In one method, an enzymatic digestion is performed by adding up to two mL of proteinase K solution (0.005% proteinase K, 30 U/mg protein, in 50 mM Tris hydroxymethyl aminomethane hydrochloride (pH 7.0), 10 mM EDTA, and 10 mM sodium chloride), with optional DNase addition, and incubating for a time period selected from the range of 1 min, 5 min, 10 min, 15 min, 30 min, 60 min, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours at 37° C. or up to 60° C. or other temperatures.

2 1000 460 If cells are to be produced, the proteinase K solution can be diluted by the addition of up to 2 mL of a solution to dissolve residual extracellular matrix such as adding formulations of a reagents or mixture of components comprised of but not limited to collagenases (e.g., collagenases type I, II, III, IV, and others), elastase, trypsin, papain, hyaluronidase, chymotrypsin, neutral protease, clostripain, caseinase, neutral protease (Dispase®), DNAse, protease XIV, RNase inhibitors, or other enzymes, biochemicals, or chemicals such as EDTA, protease inhibitors, buffers, acids, or base. In one embodiment, two mL of an enzymatic cocktail containing 1 mg/ml of Collagenase/Dispase (Roche) and 100 units/ml of Hyaluronidase (Calbiochem) in PBS/0.5 mM CaCl) are added with optional DNase addition and incubated for a time period selected from the range of 1 min, 5 min, 10 min, 15 min, 30 min, 60 min, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours at 37° C. or other temperatures. The released single cellsuspension is then pulled into the processing chamberthrough a 70 μm filter as described and removed. The released cells are then centrifuged at 300 rpm for 5 minutes, and resuspended in buffer, such as PBS or HBSS, and optionally again filtered through a 70 μm or other filter. Additional processing can then be performed as appropriate for downstream procedures.

412 1050 460 413 2 2 If nuclei are to be produced, 2 mL of nuclei isolation buffer, such as NST buffer (146 mM NaCl, 10 mM Tris base at pH 7.8, 1 mM CaCl), 21 mM MgCl, 0.05% BSA, 0.2% Nonidet P-40) can be be added to the proteinase K solution and incubated for a time period selected from the range of 1 min, 5 min, 10 min, 15 min, 30 min, 60 min, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours. The released nucleisuspension is then pulled into the processing chamberthrough a 40 μm or other filter as described and removed. The released nuclei are then centrifuged at 500 rpm for 5 minutes, and resuspended in nuclei storage buffer, and optionally again filtered through a 40 μm or other filter. Additional processing can then be performed as appropriate for downstream procedures.

200 200 200 460 19 FIGS. Another preferred embodiment of cartridgeis shown inA and B. This vertical cartridgeis designed to be injection molded and then sealed with a material such as a heat sealable plastic laminate, or laser welded, or ultrasonically welded or other means to seal cartridge. It has two processing chambersfor processing samples which facilitates improved mixing during processing steps.

19 FIGS. 120 440 425 210 200 200 100 80 50 210 2125 480 440 461 462 200 1440 Referring toA and B, a typical process flow is as follows. The operator inserts tissue specimeninto the preprocessing chamberthrough sample inlet portand places cap(not shown) onto cartridgeand inserts cartridgeinto the Singulator System, Tissue Processing System, or Sample Processing Systemas described above. After selection of the appropriate program, the instrument makes the mechanical connection to capthrough rotary motor coupler(not shown) and fluid/gas connections to the fluid/gas inlets/outlets. The instrument also contacts the preprocessing chamberand the two processing chambersandfrom the back of cartridgewith elements such as cartridge Peltierwhich can heat or cool said fluid chambers.

410 600 1430 440 480 441 1475 440 410 120 1000 1050 410 420 100 120 218 420 421 440 1000 1050 120 411 420 421 1000 410 1050 414 420 211 440 The appropriate enyzmatic or chemical dissolution solutionis moved by fluidic subsystemfrom reagent moduleinto the preprocessing chamberfrom fluid/gas inlets/outletsthrough fluid channel. The solution may be heated or cooled by the action of the temperature regulating subsystemengaged with preprocessing chamber. The enyzmatic or chemical dissolution solutioncan contain enzymes or chemicals to help dissociate the tissue specimeninto a cell suspensionor nuclei suspension. The tissue disruptor in capacting as grinder rotoris then mechanically rotated and brought up/down by the Singulator Systemwhereby tissue specimenis separated into smaller and smaller pieces by the action of the grinding features on the headwhich can be a grinder rotorand grinder statoron the bottom of preprocessing chamber. Single cellor nuclei suspensionproduction is achieved by the combined action of the grinding elements and incubation/exposure of the tissue specimento reagents, e.g., enzymes, or chemicals, or combinations of enzymes and chemicals as described herein. After the tissue disruption is sufficiently advanced, the grinder rotorcan be brought completely down until it touches the grinder statorwhereby the singulated cellsin the enzymatic dissolution solutionor nuclei suspensionin chemical dissolution solutionare pushed around and above the grinder rotorthrough the side gapbetween the rotor and the wall of preprocessing chamber.

480 2130 1000 1050 1072 440 442 450 443 461 446 444 450 461 431 480 100 All the the fluid/gas inlets/outletsare then sealed by the syringe pumpand appropriate valves and the singulated cellsor nucleisuspension, or nucleic acidsare pulled from the preprocessing chamberthrough channelto strain chamberand then through channeltowards processing chamberby applying negative pressure through channelsor. A filter in strain chamberprevents undissociated tissue, cell aggregates, and large debris from entering processing chamber. Waste chambercan containing a liquid absorbent or solid absorbent to prevent any liquid from exiting through the fluid/gas inlets/outletsand into the Singulator System.

6001 6002 1000 6100 443 6200 6001 6002 450 1000 6100 461 6200 200 6001 6002 6001 6002 461 6200 6001 6002 6200 461 442 441 6001 6002 461 6001 6002 444 19 FIG. 20 FIG. 2 To generate microtissuesor organoidsfrom a single cellsuspension, as shown in the closeup ofin, a nozzleat the end of channelcan be used to create hanging dropletswhich can generate microtissuesor organoids. This is achieved by control of the flow from strain chamberto gently pull the single cellsuspension until a droplet is formed on nozzle. Processing chambercan be prefilled with water or buffer with in some embodiments saturated with 5% COto provide hydration to hanging dropletto control evaporation. The temperature control of cartridgeby the instrument can incubate the microtissuesor organoidsat the desired temperature, for example, 37° C. The incubation can be for minutes, hours or days before harvesting of the microtissuesor organoidsby removing the seal on processing chamber, or cutting it open depending on cartridge design. In other embodiments, once the hanging droplethas formed microtissuesor organoids, the hanging dropletin processing chambercan be released by pulling vacuum or pushing fluids through channeland into channel. Once the microtissuesor organoidsare in processing chamberthe microtissuesor organoidscan be further grown by suppling sterile growth media through channelor other channels.

6001 6002 444 445 461 462 448 6001 6002 465 461 462 200 If desired, the microtissuesor organoidscan be mixed with any added reagents by applying alternative negative (and or positive) pressure to channelsandto move the sample back and forth from processing chamberto processing chamberthrough channel. If no further processing is desired, the operator can pull out the microtissuesor organoidsthrough an opening or processing chamber cap(not shown) in the top wall of processing chamberor processing chamberor by removing the seal on the surface of the cartridge.

6001 6002 200 6001 6002 444 461 6001 6002 448 910 462 6001 6002 462 6001 6002 432 447 449 432 The microtissuesor organoidscan also be processed in vertical cartridgeby use of magnetic beads for the positive selection or depletion of specific cell types, such as stem cells, or for washing the cells and/or for exchanging the buffer. The microtissuesor organoidscan be further processed by using cell-specific affinity reagents coupled to magnetic beads. For example, cell-type specific affinity magnetic beads and reaction solutions are injected through channelinto processing chamber. The beads are incubated with the microtissuesor organoidsby mixing though channelas described above, whereby the magnetic beads bind to their target cells. Then, magnet(s)is/are applied to the frontside of processing chambers, whereby the magnetic beads (and attached microtissuesor organoids) are attracted to and held at the processing chamberwall. The microtissuesor organoidsthat does not contain the specific targets is pulled into the waste chamberby applying negative pressure to through channelsand. Waste chamberwhich can optionally contain a liquid or solid absorbent substance.

444 462 446 910 432 Simultaneously or subsequently, washing solution can be injected through channeland pulled into chamberby applying vacuum on channelto wash the beads attached to magnetby combinations of mixing, magnetic release/application and pulling liquid to the waste chamberas described. This process can be repeated one or more times.

6001 6002 910 448 6001 6002 461 462 6001 6002 461 462 465 After the microtissuesor organoidsare in the desired output media, the magnetis released, the cells homogeneously resuspended by mixing by moving the cells back and forth through channel, and then the microtissuesor organoidsis pulled either into processing chamberor. The operator can then pull out the microtissuesor organoidsthrough an opening in the top wall of Processing Chamberorcovered by a foil-seal, or septum, or processing chamber capor other mechanism (not shown). Other processing/reaction/fluidic elements can be added to the cartridge as desired to enable additional processing modes in including without limitation tangential flow filtration, optical interrogation, library preparation, and nucleic acid purification.

6001 6002 1000 1050 6001 6002 500 6001 6002 200 Similar processing methods can also be used to resuspend the microtissuesor organoidsin a specific media, buffer, or growth solution, such as Matricel, or to perform labeling with chemicals such as mass tags, or fluorescent dyes, or Raman labels, or other labels. In addition, similar methods can be used to chemically or biochemically modify single cellsor nucleior microtissuesor organoidsincluding screening of potential therapeutic compounds, or inhibitors of growth or viability. In some embodiments, Measurement Subsystemcan interrogate the microtissuesor organoidsinside cartridge.

1000 461 1000 461 In another embodiment, a single cellsuspension is pulled directly into processing chamberand stem cells purified by magnetic bead processing as described with non-stem cells removed. In another embodiment, a single cellsuspension is pulled directly into processing chamberand chemically induced into stem cells, or with transcription factors, or by by retroviral-mediated expression of the four transcription factors Oct4, Sox2, cMyc, Klf4. Many other modalities are possible.

The degradation of RNA in nuclei during and after nuclei isolation can alter the amount and representation of RNA. The degradation is tissue specific and currently can prevent single nuclei sequencing of the transcriptome from tissues with high RNAse activity such as pancreas. Similarly, RNA or other biomolecules from other subcellular components such as nuclei and mitochondria can be degraded during isolation procedures. A method to improve the quality of RNA and other biomolecules comprised of proteins, lipids, polysaccharides, etc. isolated from solid tissue samples is described.

Current methods to dissociate solid tissues into nuclei, using reagents alone or in combination with mechanical disruption techniques, can result in RNA becoming severely degraded and therefore not useful for downstream genomic analyses. Current methods to preserve RNA quality include the use of high concentrations of RNAse inhibitors, performing operations at low temperature, and performing operations quickly. The action of RNAses on RNA within a nucleus are rapid enzymatic reactions. Addition of RNAse inhibitors that bind to RNAses can be ineffective for tissue types that exhibit high levels of RNAse activity. Performing operations at 4° C. can lower the rate of enzymatic activity, but again, if there are high levels of RNAses in the tissue sample, simply lowering the temperature, even in the presence of RNAse inhibitors, can fail to adequately protect RNA from degradation. Isolating nuclei from solid tissue samples in the 1-1000 mg range may also require total reagent volumes of 0.5 to 5 ml, and including RNAse inhibitor reagents at the typical one unit/microliter concentration can cost hundreds of dollars per sample.

This instant invention describes the use of additives to reduce the rate of degradation. In one method, proteinase K, a serine protease, or other proteases are added to degrade RNAses or DNases released from the extracellular matrix or upon lysis of cell membranes.

In another embodiment, reagents to increase the viscosity are added during the isolation of nuclei or other subcellular components, thereby reducing the rate of diffusion of DNases, RNAses, lipases, nucleases, proteases, and other degradatory enzymes, and therefore reducing the level of RNA degradation or other biomolecule degradation during the isolation procedure for nuclei, mitochondria or other subcellular components. Examples of such additives include, but aren't limited to, crowding agents, and biocompatible high molecular weight polymers comprised of ficoll, dextran, sucrose, trehalose, cellulose, and polyethylene glycol. Typical concentrations of such reagents used are approximately but not limited to 0.01% to 50% w/v.

412 413 100 412 413 A preferred embodiment of the method applied to isolating nuclei from solid tissues or previously prepared single cell suspensions is to include one or more of the additives in either a nuclei isolation solution, nuclei storage solution, or both as used to isolate nuclei from tissue samples. For example, when using the Singulator Systemfor isolating nuclei, the nuclei isolation solutionmight have 5% w/v ficoll added or the nuclei storage solutionmight have 5% w/v ficoll. One or both of these solutions might also contain one or more protease inhibitors, and one or more RNAse inhibitor reagents including but not limited to SUPERase·ln RNase Inhibitor, RNaseOUT Recombinant Ribonuclease Inhibitor, RNAsecure RNase Inactivation Reagent, Recombinant RNase Inhibitor and small molecule reagents including, but not limited to nucleotides and inorganic phosphates.

412 413 1430 1) Loading the nuclei isolation solutionand nuclei storage solutionwith additives to increase viscosity onto the reagent module. 120 200 210 2) Placing a fresh or flash frozen mouse kidney tissue specimenin a cartridgeprecooled at 4° C. and adding tissue disruptor cap. 200 2050 3) Placing cartridgein a Single Sample Singulator instrumentset to 4° C. operating temperature. 740 412 120 440 460 413 440 413 460 4) Selecting the nuclei isolation protocol from the software user interfaceand selecting “Run”. The Singulator then delivers 2 mL of the nuclei isolation solutionto the mouse kidney tissue specimenin the preprocessing chamber; mechanically disrupts the tissue at 135 rpm; pulls the sample through a 40 micron strainer into the processing chamber; adds 2 mL of the nuclei storage solutionto preprocessing chamberto rinse residual material and decrease the final detergent concentration to quench disruption; pulls the added nuclei storage solutionthrough the filter into the processing chamber. 200 2050 1050 413 413 5) The sample cartridgeis then removed from the Single Sample Singulator instrument, the nucleisuspension pipetted into a 5 ml tube, and 2 mL of 4° C. nuclei storage bufferadded. The sample is then centrifuged at 4° C. for 5 minutes at 500 g. The supernatant is pipetted out and discarded. The nuclei pellet is then resuspended in one mL of nuclei storage buffer. A protocol for improved isolation of mouse kidney nuclei from 300 mg of fresh or flash frozen mouse kidney tissue might be comprised of:

Disrupting intact tissue into single cells can induce transcriptional changes in the cells, through a process known as anoikis or other stress-response pathways. Such changes can lead to cell death or confound later genomic or proteomic analyses. Use of quantitative PCR (qPCR assays) on a panel of targeted genes known to be involved in anoikis or other cell-stress pathways can be used to characterize the dissociation-related transcriptional changes in the single cells produced by dissociation. The qPCR data can also be used to inform and optimize the dissociation process to reduce the stress-induced changes. While panels of genes have been described for monitoring specific cell stress pathways, none have been created to inform anoikis-induced stress or stress resulting from mechanical and/or enzymatic/chemical tissue disruption.

qPCR panels can be used to identify specific cell types or sub-cell types that are present in a mixture of dissociated cells or characterize individual cells that have been isolated. The cell identity information can in turn be used to inform and optimize the dissociation process for desired cell types.

The panel may also be used to characterize RNA isolated from nuclei as opposed to single cells. Processes for isolating nuclei can be much faster than for isolating cells. The shorter process time may reduce the amount of cell stress evident in the gene expression data. In addition, isolated nuclei will lack RNA from the cell cytoplasm and will therefore provide complementary data. The qPCR data can also be used to inform and optimize the dissociation process to reduce the stress-induced changes or to identify specific cell types of origin for nuclei.

The structure of an exemplary panel for cell stress shown in Table 1 is a collection of PCR primers chosen to amplify genes associated with cell stress, and that have been optimized to amplify RNA sequences rather than genomic DNA. The panels can consists of 1 to over 200 genes, and may include at least one housekeeping gene used as an internal control.

100 To use the panel, after isolation of cells from solid tissue using a device such as, but not limited to, the Singulator System, a user would perform RT-qPCR on a panel of genes involved in cell stress responses or cellular identity on a known number of cells, or nuclei, or known quantity of isolated RNA. The levels of gene expression would be determined and may be (1) compared to the level of expression of so-called housekeeping genes whose expression is not affected by cell stress responses, (2) compared to the level of gene expression obtained from cells isolated using a different isolation protocol, or (3) used to identify the presence or absence or specific cell types. Other analyses are also possible.

One example of a gene panel is shown below, with 38 genes suspected of being involved in cellular stress responses and two housekeeping genes used as internal controls. The genes have been chosen because they are broadly expressed in most tissues, enabling the panel to be used with cells derived from a variety of tissue samples. The panel shown in Table 1 was developed for use with mouse tissues. Genes marked with an asterix are the control housekeeping genes.

100 1000 200 As an example, a user would disrupt a fresh mouse liver sample into a suspension of cells using the Singulator Systemand a protocol for mouse liver. The user would then remove the cell suspensionfrom the Singulator sample cartridge. After using a Countess, hemocytometer, FACS, or similar method to determine cell concentration, the user could employ a “Cells to Ct” kit (Invitrogen), or alternative method for performing cell lysis, cDNA synthesis and qPCR with the primer sets for the genes listed in the panel. Alternatively, the user could purify RNA from the isolated cells using an RNA isolation kit, e.g., RNA Easy kit (Qiagen), or alternatively, quantify the RNA concentration and purity, then perform a cDNA synthesis and qPCR experiment using the panel of genes. The qPCR amplification would be run on a Real Time PCR instrument with a thermal cycling profile appropriate to the kit or methods used. The qPCR experiment will return a cycle threshold (Ct) values for each gene and these data can be used to assess relative gene expression patterns.

Other assays for cell stress responses are available, including those based on measuring apoptosis or necrosis of cells. The panel defined in Table 1 is unique in that it represents genes that span several cell stress pathways, whether known or uncharacterized, and can be used to measure cell stress responses specifically to a tissue disruption process. It is designed to encompass an array of genes that may respond to tissue dissociation, which may trigger multiple stress pathways, rather than monitor one or a limited number of defined stress genes or pathways.

The use of CRISPR, TALENS, and other gene editing techniques are being increasingly used to experimentally manipulate biological systems for both research and clinical applications. Key metrics for the success or failure of such manipulations are the number of cells with effectively altered genomes and the specificity of such alterations at the desired locations as opposed to off-target editing. Off target gene editing can lead to disease-causing changes to cells. It is difficult to assess the penetrance of editing or the specificity through DNA or RNA sequencing of bulk tissue, as rare events may not be observable. It is important to perform an evaluation of gene editing using single cell sequencing techniques.

120 6001 6002 100 In this example, a biological test subject, such as but not limited to cell cultures, adherent cells, organoids, model organisms, or human patients has been treated with a gene editing process. Subsequent to the treatment, a sample or samples of cells or tissue is removed from the test organism or culture media. The sample, such as a tissue specimenor microtissueor organoid, is processed in the Singulatoror alternative device for tissue disruption to obtain a suspension of cells or nuclei. The cells or nuclei are then subjected to single cell or single nuclei DNA or RNA sequencing to determine the presence or absence of an edited genome and the representation within the single cell population.

As used herein, the following meanings apply unless otherwise specified. The word “may” is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). The words “include”, “including”, and “includes” and the like mean including, but not limited to. The singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “an element” includes a combination of two or more elements, notwithstanding use of other terms and phrases for one or more elements, such as “one or more.” The term “or” is, unless indicated otherwise, non-exclusive, i.e., encompassing both “and” and “or.”

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inherent to such process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive-or and not to an exclusive-or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present). Both plural and singular means may be included. The term “any of” between a modifier and a sequence means that the modifier modifies each member of the sequence. So, for example, the phrase “at least any of 1, 2 or 3” means “at least 1, at least 2 or at least 3”. The term “consisting essentially of” refers to the inclusion of recited elements and other elements that do not materially affect the basic and novel characteristics of a claimed combination.

All patents, patent applications, published applications, treatises and other publications referred to herein, both supra and infra, are incorporated by reference in their entirety. If a definition and/or description is set forth herein that is contrary to or otherwise inconsistent with any definition set forth in the patents, patent applications, published applications, and other publications that are herein incorporated by reference, the definition and/or description set forth herein prevails over the definition that is incorporated by reference.

It should be understood that the description and the drawings are not intended to limit the invention to the particular form disclosed, but to the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description and the drawings are to be construed as illustrative only and are for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed or omitted, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims. Headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description.